Rna interference in ocular indications

ABSTRACT

The present invention relates to ocular administration of sd-rxRNA and rxRNAori molecules.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Serial No. U.S. 61/317,254, entitled “RNAINTERFERENCE IN OCULAR INDICATIONS,” filed on Mar. 24, 2010, and U.S.Provisional Application Serial No. U.S. 61/317,621, entitled “RNAINTERFERENCE IN OCULAR INDICATIONS,” filed on Mar. 25, 2010, the entiredisclosures of which are herein incorporated by reference in theirentireties.

FIELD OF INVENTION

The invention pertains to the field of RNA interference (RNAi). Theinvention more specifically relates to ocular administration of nucleicacid molecules with improved in vivo delivery properties and their usein efficient gene silencing.

BACKGROUND OF INVENTION

Complementary oligonucleotide sequences are promising therapeutic agentsand useful research tools in elucidating gene functions. However, priorart oligonucleotide molecules suffer from several problems that mayimpede their clinical development, and frequently make it difficult toachieve intended efficient inhibition of gene expression (includingprotein synthesis) using such compositions in vivo.

A major problem has been the delivery of these compounds to cells andtissues. Conventional double-stranded RNAi compounds, 19-29 bases long,form a highly negatively-charged rigid helix of approximately 1.5 by10-15 nm in size. This rod type molecule cannot get through thecell-membrane and as a result has very limited efficacy both in vitroand in vivo. As a result, all conventional RNAi compounds require somekind of a delivery vehicle to promote their tissue distribution andcellular uptake. This is considered to be a major limitation of the RNAitechnology.

There have been previous attempts to apply chemical modifications tooligonucleotides to improve their cellular uptake properties. One suchmodification was the attachment of a cholesterol molecule to theoligonucleotide. A first report on this approach was by Letsinger etal., in 1989. Subsequently, ISIS Pharmaceuticals, Inc. (Carlsbad,Calif.) reported on more advanced techniques in attaching thecholesterol molecule to the oligonucleotide (Manoharan, 1992).

With the discovery of siRNAs in the late nineties, similar types ofmodifications were attempted on these molecules to enhance theirdelivery profiles. Cholesterol molecules conjugated to slightly modified(Soutschek, 2004) and heavily modified (Wolfrum, 2007) siRNAs appearedin the literature. Yamada et al., 2008 also reported on the use ofadvanced linker chemistries which further improved cholesterol mediateduptake of siRNAs. In spite of all this effort, the uptake of these typesof compounds appears to be inhibited in the presence of biologicalfluids resulting in highly limited efficacy in gene silencing in vivo,limiting the applicability of these compounds in a clinical setting.

SUMMARY OF INVENTION

Described herein are methods and compositions for efficient in vivoadministration of sd-rxRNA molecules to the eye. Surprisingly,intraocular (e.g., intravitreal and subretinal) administration ofsd-rxRNA molecules resulted in distribution and uptake of the sd-rxRNAby all the cell layers in the retina. These molecules have widespreadapplications for treatment of disorders or conditions associated withthe eye.

Aspects of the invention relate to methods for delivering a nucleic acidto an eye of a subject in need thereof, comprising administering to theeye of the subject an sd-rxRNA, in an effective amount to promote RNAinterference by the sd-rxRNA in the eye. In some embodiments, theadministration of the sd-rxRNA is intravitreal.

In some embodiments, the method is for treating an ocular disorder. Incertain embodiments, the ocular disorder is vascular leakage,neovascularization, age-related macular degeneration (AMD), choroidalneovascularization (wet AMD), geographic atrophy (advanced dry AMD),early-to-intermediate dry AMD, post surgical cystoid macular edema(CME), nonproliferative diabetic retinopathy (NPDR), diabetic macularedema (DME), macular edema secondary to retinal vein occlusion (RVO),proliferative diabetic retinopathy (PDR), glaucoma, neovascular glaucoma(NVG), retinopathy of prematurity (ROP), fibroproliferative retinaldisease, proliferative vitreoretinopathy (PVR), epiretinalmembranes/vitreomacular adhesions, retinal degenerative disease,retinitis pigmentosa, retinal vascular occlusive disorders, retinal veinocclusion, retinal artery occlusion, retinoblastoma, trabeculectomyfailure due to scarring, or uveitis. In one embodiment, the oculardisorder is AMD. In another embodiment, the ocular disorder is DME. Inyet another embodiment, the ocular disorder is PVR. In still anotherembodiment, the ocular disorder is trabeculectomy failure due toscarring.

In certain embodiments, the sd-rxRNA is directed against a gene encodingfor VEGF, MAP4K4, PDGF-B, SDF-1, IGTA5, ANG2, CTGF, HIF-1α, mTOR, SDF-1,PDGF-B, SPP1, PTGS2 (COX-2), TGFβ1, TGFβ2, complement factors 3 or 5,PDGFRα, PPIB, or myc, or a combination thereof.

In some embodiments, the sd-rxRNA is directed against a gene encodingfor VEGF. In certain embodiments, the sd-rxRNA is directed against asequence selected from the sequences within Table 2. In one embodiment,the sd-rxRNA is directed against a sequence comprising at least 12contiguous nucleotides of a sequence selected from the sequences withinTable 2. In another embodiment the sd-rxRNA comprises at least 12contiguous nucleotides of a sequence selected from the sequences withinTables 3-8 or 10. In yet another embodiment, the sense strand of thesd-rxRNA comprises at least 12 contiguous nucleotides of the sequence ofSEQ ID NO:1317 or SEQ ID NO:1357. In still another embodiments, theantisense strand of the sd-rxRNA comprises at least 12 contiguousnucleotides of the sequence of SEQ ID NO:1318 or SEQ ID NO:1358. In afurther embodiment, the sense strand of the sd-rxRNA comprises SEQ IDNO:1317 and the antisense strand of the sd-rxRNA comprises SEQ IDNO:1318. In one embodiment, the sense strand of the sd-rxRNA comprisesSEQ ID NO:1357 and the antisense strand of the sd-rxRNA comprises SEQ IDNO:1358. In another embodiment, the sense strand of the sd-rxRNAcomprises SEQ ID NO:1379 and the antisense strand of the sd-rxRNAcomprises SEQ ID NO:1380. In yet another embodiment, the sense strand ofthe sd-rxRNA comprises SEQ ID NO:1397 and the antisense strand of thesd-rxRNA comprises SEQ ID NO:1398.

In certain embodiments, the sd-rxRNA is directed against a gene encodingfor CTGF. In one embodiment, the antisense strand of the sd-rxRNAcomprises at least 12 contiguous nucleotides of the sequence of SEQ IDNO:948 or SEQ ID NO:964. In another embodiment, the sense strand of thesd-rxRNA comprises at least 12 contiguous nucleotides of the sequence ofSEQ ID NO:947 or SEQ ID NO:963.

In some embodiments, two or more different sd-rxRNAs that are directedagainst genes encoding for two or more different proteins are bothadministered to the eye of the subject. In one embodiment, the sd-rxRNAsare directed against VEGF and CTGF. In another embodiment, the sd-rxRNAsare directed against VEGF and PTGS2 (COX-2).

In certain aspects, the sd-rxRNA of any one of the foregoing embodimentsis hydrophobically modified. In some embodiments, the sd-rxRNA is linkedto one or more hydrophobic conjugates.

In certain aspects, the sd-rxRNA of any one of the foregoingembodiments, includes at least one 5-methyl C or U modifications.

Other aspects of the invention relate to methods for delivering anucleic acid to an eye of a subject in need thereof, includingadministering to the eye of the subject an rxRNAori, in an effectiveamount to promote RNA interference by the rxRNAori in the eye. In oneembodiment, the rxRNAori is directed against VEGF. In anotherembodiment, the rxRNAori is directed against a sequence comprising atleast 12 contiguous nucleotides of a sequence selected from thesequences within Table 2. In yet another embodiment, the sense strand ofthe rxRNAori comprises at least 12 contiguous nucleotides of thesequence of SEQ ID NO:13 or SEQ ID NO:28. In still another embodiment,the antisense strand of the rxRNAori comprises at least 12 contiguousnucleotides of the sequence of SEQ ID NO:1377 or SEQ ID NO:1378.

Some aspects of the invention relate to sd-rxRNAs directed against asequence selected from the sequences within Table 2. Other aspectsrelate to sd-rxRNAs directed against a sequence comprising at least 12contiguous nucleotides of a sequence selected from the sequences withinTable 2. Yet other aspects relate to sd-rxRNAs that comprises at least12 contiguous nucleotides of a sequence selected from the sequenceswithin Tables 3-8 or 10. In some embodiments, the sense strand of thesd-rxRNA comprises at least 12 contiguous nucleotides of the sequence ofSEQ ID NO:1317, 1357, 1379 or 1397. In other embodiments, the antisensestrand of the sd-rxRNA comprises at least 12 contiguous nucleotides ofthe sequence of SEQ ID NO:1318, 1358, 1380 or 1398. In yet otherembodiments, the antisense strand of the sd-rxRNA comprises SEQ IDNO:1318. In still other embodiments, the sense strand of the sd-rxRNAcomprises SEQ ID NO:1357 and the antisense strand of the sd-rxRNAcomprises SEQ ID NO:1358. In further embodiments, the sense strand ofthe sd-rxRNA comprises SEQ ID NO:1379 and the antisense strand of thesd-rxRNA comprises SEQ ID NO:1380. In still further embodiments, thesense strand of the sd-rxRNA comprises SEQ ID NO:1397 and the antisensestrand of the sd-rxRNA comprises SEQ ID NO: 1398. In certainembodiments, the sd-rxRNA is hydrophobically modified. In otherembodiments, the sd-rxRNA is linked to one or more hydrophobicconjugates. In certain other embodiments, the sd-rxRNA includes at leastone 5-methyl C or U modifications.

Other aspects of the invention relate to compositions comprising ansd-rxRNA of any one of any one of the foregoing aspects or embodiments.In one embodiment, the composition further comprises an sd-rxRNA that isdirected against a gene encoding for a protein other than VEGF. Inanother embodiment, the composition comprises an sd-rxRNA that isdirected against a gene encoding for CTGF and/or PTGS2 (COX-2).

Aspects of the invention also relate to an rxRNAori that is directedagainst a sequence selected from the sequences within Table 2. Anotheraspect relates to an rxRNAori that is directed against a sequencecomprising at least 12 contiguous nucleotides of a sequence selectedfrom the sequences within Table 2. In one embodiment, the sense strandof the rxRNAori comprises at least 12 contiguous nucleotides of thesequence of SEQ ID NO:13 or SEQ ID NO:28. In another embodiment, theantisense strand of the rxRNAori comprises at least 12 contiguousnucleotides of the sequence of SEQ ID NO:1377 or SEQ ID NO:1378.

Another aspect of the invention relates to a composition comprising anrxRNAori of any one of the foregoing aspects or embodiments. In oneembodiment, the composition comprises an rxRNAori that is directedagainst a gene encoding for a protein other than VEGF. In anotherembodiment, the composition comprises an rxRNAori that is directedagainst a gene encoding for CTGF and/or PTGS2 (COX-2).

In some embodiments, the sd-rxRNA within the composition ishydrophobically modified. In certain embodiments, the sd-rxRNA is linkedto one or more hydrophobic conjugates. In some embodiments, the sd-rxRNAis linked to a lipophilic group. In certain embodiments, the lipophilicgroup is linked to the passenger strand of the sd-rxRNA. In someembodiments, the sd-rxRNA is linked to cholesterol, a long chain alkylcholesterol analog, vitamin A, or vitamin E. In some embodiments, thesd-rxRNA is attached to chloroformate.

The sd-rxRNA can include at least one 2′ O methyl or 2′ fluoromodification and/or at least one 5-methyl C or U modification. In someembodiments, the sd-rxRNA has a guide strand of 16-28 nucleotides inlength. In certain embodiments, at least 40% of the nucleotides of thesd-rxRNA are modified. The sd-rxRNA can be attached to a linker which incertain embodiments is protonatable.

In some embodiments, the sd-rxRNA contains at least two single strandedregions which can contain phosphorothioate modifications. In certainembodiments, the single stranded regions are located at the 3′ end ofthe guide strand and the 5′ end of the passenger strand.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 presents a confocal triple overlay of DIC, DY547 and Hoechstindicating that by 24 hours after intravitreal or subretinal dosing,sd-rxRNA has penetrated the entire retina.

FIG. 2 demonstrates that sd-rxRNA have improved retinal delivery overrxRNAori.

Top panels represent images taken immediately post-dose, indicatingintravitreal delivery of sd-rxRNA and rxRNAori. Middle and bottom panelspresent fluorescent images taken 24 and 48 hours, respectively, afterdosing, demonstrating retinal delivery of only sd-rxRNA.

FIG. 3 demonstrates that a fluorescent signal is detected throughout theretina following intravitreal dosing of sd-rxRNA, but not followingdosing of PBS or rxRNAori.

FIG. 4 demonstrates that sd-rxRNA penetrates the retina to the outersegments of the photoreceptors.

FIG. 5 demonstrates robust uptake of and silencing by sd-rxRNA inARPE-19 cells, as compared to uptake of and silencing by rxRNAori inARPE-19 cells.

FIG. 6 demonstrates non-limiting examples of sd-rxRNA having ocularpotential.

The optimized lead molecules in FIG. 6 correspond to the following SEQID NOs, representing the sense and antisense strands, respectively: lead21212: SEQ ID NOs 963 and 964; lead 21214: SEQ ID NOs 967 and 968; lead21215: SEQ ID NOs 969 and 970; lead 21204: SEQ ID NOs 947 and 948; lead21205: SEQ ID NOs 949 and 950; lead 21227: SEQ ID NOs 993 and 994; lead21381: SEQ ID NOs 1011 and 1012; lead 21382: SEQ ID NOs 1013 and 1014;lead 21429: SEQ ID NOs 1303 and 1024; lead 21430: SEQ ID NOs 1025 and1026; lead 21383: SEQ ID NOs 1015 and 1016; lead 21224: SEQ ID NOs 987and 988; lead 21228: SEQ ID NOs 1399 and 1400; lead 21229: SEQ ID NOs1401 and 1402; lead 21230: SEQ ID NOs 1403 and 1404; lead 21393: SEQ IDNOs 1405 and 1406; lead 21394: SEQ ID NOs 1407 and 1408; lead 21233: SEQID NOs 1409 and 1410; lead 21234: SEQ ID NOs 1411 and 1412; lead 21360:SEQ ID NOs 1413 and 1414; lead 21374: SEQ ID NOs 1304 and 1314; lead21366: SEQ ID NOs 1415 and 1416; lead 21368: SEQ ID NOs 1417 and 1418;lead 21379: SEQ ID NOs 1155 and 1156; lead 21380: SEQ ID NOs 1157 and1158; lead 21352: SEQ ID NOs 1419 and 1420; and lead 21777: SEQ IDNO:1421.

FIG. 7 demonstrates identification of VEGF sd-rxRNA.

FIG. 8 demonstrates progression of sd-rxRNA penetration to cell layersof the eye after intravitreal dosing. sd-rxRNA penetrates the ganglioncell layer by 2-3 hours post dose. sd-rxRNA penetrates to the retinalpigment epithelium (RPE) and outer segments of the photoreceptors by 24hours post dose.

FIG. 9 demonstrates by confocal microscopy that while sd-rxRNApenetrates to the RPE and outer segments of the photoreceptors, there isno visible penetration of convention RNAi compounds.

FIG. 10 demonstrates that fluorescent sd-rxRNA, but not rxRNAori, can bedetected in all rabbit retinal cell layers, 24 hours post dose.

FIG. 11 presents confocal images at high magnification, demonstratingthat sd-rxRNA penetrates all rabbit retinal cell layers 24 hours postdose.

FIG. 12 demonstrates dose-dependent silencing in vivo by sd-rxRNA, afterintravitreal administration.

FIG. 13 demonstrates the duration of PPIB silencing by sd-rxRNA in themouse eye.

FIG. 14 demonstrates the duration of MAP4K4 silencing by sd-rxRNA in themouse eye.

FIG. 15 demonstrates that sd-rxRNA does not induce blood vessel leakagethree weeks post dose.

FIG. 16 demonstrates that sd-rxRNA does not induce architectural damagethree weeks post dose.

FIG. 17 reveals that sd-rxRNA does not impair retinal function 3 weekspost dose.

FIG. 18 demonstrates that multiple sd-rxRNA constructs can be used totarget multiple genes simultaneously.

FIG. 19 demonstrates that variation of linker chemistry does notinfluence silencing activity of sd-rxRNAs in vitro. Two different linkerchemistries were evaluated, a hydroxyproline linker and ribo linker, onmultiple sd-rxRNAs (targeting Map4k4 or PPIB) in passive uptake assaysto determine linkers which favor self delivery. HeLa cells weretransfected in the absence of a delivery vehicle (passive transfection)with sd-rxRNAs at 1 uM, 0.1 uM or 0.01 uM for 48 hrs. Use of eitherlinker results in an efficacious delivery of sd-rxRNA.

DETAILED DESCRIPTION

Aspects of the invention relate to methods and compositions involved ingene silencing. The invention is based at least in part on thesurprising discovery that delivery to the eye, including subretinal andintravitreal injection of sd-rxRNAs results in efficient distributionand uptake by all cell layers in the retina, including the retinalpigment epithelium layer. Drastically better retinal uptake anddistribution is observed for sd-rxRNAs than for conventional RNAicompounds. Thus, sd-rxRNAs represent a new class of therapeutic RNAimolecules with significant potential in treatment of ocular conditionsor disorders.

sd-rxRNA Molecules

Aspects of the invention relate to sd-rxRNA molecules. As used herein,an “sd-rxRNA” or an “sd-rxRNA molecule” refers to a self-delivering RNAmolecule such as those described in, and incorporated by reference from,PCT Publication No. WO2010/033247 (Application No. PCT/US2009/005247),filed on Sep. 22, 2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAICOMPOUNDS.” Briefly, an sd-rxRNA, (also referred to as ansd-rxRNA^(nano)) is an isolated asymmetric double stranded nucleic acidmolecule comprising a guide strand, with a minimal length of 16nucleotides, and a passenger strand of 8-18 nucleotides in length,wherein the double stranded nucleic acid molecule has a double strandedregion and a single stranded region, the single stranded region having4-12 nucleotides in length and having at least three nucleotide backbonemodifications. In preferred embodiments, the double stranded nucleicacid molecule has one end that is blunt or includes a one or twonucleotide overhang. sd-rxRNA molecules can be optimized throughchemical modification, and in some instances through attachment ofhydrophobic conjugates.

In some embodiments, an sd-rxRNA comprises an isolated double strandednucleic acid molecule comprising a guide strand and a passenger strand,wherein the region of the molecule that is double stranded is from 8-15nucleotides long, wherein the guide strand contains a single strandedregion that is 4-12 nucleotides long, wherein the single stranded regionof the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12phosphorothioate modifications, and wherein at least 40% of thenucleotides of the double stranded nucleic acid are modified.

The polynucleotides of the invention are referred to herein as isolateddouble stranded or duplex nucleic acids, oligonucleotides orpolynucleotides, nano molecules, nano RNA, sd-rxRNA^(nano), sd-rxRNA orRNA molecules of the invention.

sd-rxRNAs are much more effectively taken up by cells compared toconventional siRNAs. These molecules are highly efficient in silencingof target gene expression and offer significant advantages overpreviously described RNAi molecules including high activity in thepresence of serum, efficient self delivery, compatibility with a widevariety of linkers, and reduced presence or complete absence of chemicalmodifications that are associated with toxicity.

In contrast to single-stranded polynucleotides, duplex polynucleotideshave traditionally been difficult to deliver to a cell as they haverigid structures and a large number of negative charges which makesmembrane transfer difficult. sd-rxRNAs however, although partiallydouble-stranded, are recognized in vivo as single-stranded and, as such,are capable of efficiently being delivered across cell membranes. As aresult the polynucleotides of the invention are capable in manyinstances of self delivery. Thus, the polynucleotides of the inventionmay be formulated in a manner similar to conventional RNAi agents orthey may be delivered to the cell or subject alone (or with non-deliverytype carriers) and allowed to self deliver. In one embodiment of thepresent invention, self delivering asymmetric double-stranded RNAmolecules are provided in which one portion of the molecule resembles aconventional RNA duplex and a second portion of the molecule is singlestranded.

The oligonucleotides of the invention in some aspects have a combinationof asymmetric structures including a double stranded region and a singlestranded region of 5 nucleotides or longer, specific chemicalmodification patterns and are conjugated to lipophilic or hydrophobicmolecules. This class of RNAi like compounds have superior efficacy invitro and in vivo. It is believed that the reduction in the size of therigid duplex region in combination with phosphorothioate modificationsapplied to a single stranded region contribute to the observed superiorefficacy.

The invention is based, at least in part, on the surprising discoverythat sd-rxRNAs can be delivered efficiently to the eye through eithersubretinal or intravitreal injection. Based on results generated inmultiple different mammalian systems, including mouse, rat and rabbit,and as presented in the Examples section, drastically (several orders ofmagnitude) better ocular uptake and distribution is observed followingadministration of sd-rxRNAs than following administration ofconventional RNAi compounds.

Another surprising aspect of the invention is that sd-rxRNA moleculesare taken up by all cell layers in the retina, including the retinalpigment epithelium cell layer. Efficient sd-rxRNA distribution isachieved through both subretinal and intravitreal injection and bothmeans of administration are compatible with aspects of the invention. Insome embodiments, intravitreal administration is preferred due totechnical ease and widespread use in intraocular drug delivery.

As used herein, “ocular” refers to the eye, including any and all of itscells including muscles, nerves, blood vessels, tear ducts, membranesetc., as well as structures that are connected with the eye and itsphysiological functions. The terms ocular and eye are usedinterchangeably throughout this disclosure. Non-limiting examples ofcell types within the eye include: cells located in the ganglion celllayer (GCL), the inner plexiform layer inner (IPL), the inner nuclearlayer (INL), the outer plexiform layer (OPL), outer nuclear layer (ONL),outer segments (OS) of rods and cones, the retinal pigmented epithelium(RPE), the inner segments (IS) of rods and cones, the epithelium of theconjunctiva, the iris, the ciliary body, the corneum, and epithelium ofocular sebaceous glands.

In a preferred embodiment the RNAi compounds of the invention comprisean asymmetric compound comprising a duplex region (required forefficient RISC entry of 8-15 bases long) and single stranded region of4-12 nucleotides long. In some embodiments, the duplex region is 13 or14 nucleotides long. A 6 or 7 nucleotide single stranded region ispreferred in some embodiments. The single stranded region of the newRNAi compounds also comprises 2-12 phosphorothioate internucleotidelinkages (referred to as phosphorothioate modifications). 6-8phosphorothioate internucleotide linkages are preferred in someembodiments. Additionally, the RNAi compounds of the invention alsoinclude a unique chemical modification pattern, which provides stabilityand is compatible with RISC entry. The combination of these elements hasresulted in unexpected properties which are highly useful for deliveryof RNAi reagents in vitro and in vivo.

The chemical modification pattern, which provides stability and iscompatible with RISC entry includes modifications to the sense, orpassenger, strand as well as the antisense, or guide, strand. Forinstance the passenger strand can be modified with any chemical entitieswhich confirm stability and do not interfere with activity. Suchmodifications include 2′ ribo modifications (O-methyl, 2′ F, 2 deoxy andothers) and backbone modification like phosphorothioate modifications. Apreferred chemical modification pattern in the passenger strand includesOmethyl modification of C and U nucleotides within the passenger strandor alternatively the passenger strand may be completely Omethylmodified. The guide strand, for example, may also be modified by anychemical modification which confirms stability without interfering withRISC entry. A preferred chemical modification pattern in the guidestrand includes the majority of C and U nucleotides being 2′ F modifiedand the 5′ end being phosphorylated. Another preferred chemicalmodification pattern in the guide strand includes 2′Omethyl modificationof position 1 and C/U in positions 11-18 and 5′ end chemicalphosphorylation. Yet another preferred chemical modification pattern inthe guide strand includes 2′ Omethyl modification of position 1 and C/Uin positions 11-18 and 5′ end chemical phosphorylation and 2′Fmodification of C/U in positions 2-10. In some embodiments the passengerstrand and/or the guide strand contains at least one 5-methyl C or Umodifications.

In some embodiments, at least 30% of the nucleotides in the sd-rxRNA aremodified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the sd-rxRNA aremodified. In some embodiments, 100% of the nucleotides in the sd-rxRNAare modified.

The above-described chemical modification patterns of theoligonucleotides of the invention are well tolerated and actuallyimproved efficacy of asymmetric RNAi compounds. It was also demonstratedexperimentally herein that the combination of modifications to RNAi whenused together in a polynucleotide results in the achievement of optimalefficacy in passive uptake of the RNAi. Elimination of any of thedescribed components (Guide strand stabilization, phosphorothioatestretch, sense strand stabilization and hydrophobic conjugate) orincrease in size in some instances results in sub-optimal efficacy andin some instances complete lost of efficacy. The combination of elementsresults in development of a compound, which is fully active followingpassive delivery to cells such as HeLa cells. The data in the Examplespresented below demonstrates high efficacy of the oligonucleotides ofthe invention in vivo upon ocular administration.

The sd-rxRNA can be further improved in some instances by improving thehydrophobicity of compounds using of novel types of chemistries. Forexample, one chemistry is related to use of hydrophobic basemodifications. Any base in any position might be modified, as long asmodification results in an increase of the partition coefficient of thebase. The preferred locations for modification chemistries are positions4 and 5 of the pyrimidines. The major advantage of these positions is(a) ease of synthesis and (b) lack of interference with base-pairing andA form helix formation, which are essential for RISC complex loading andtarget recognition. A version of sd-rxRNA compounds where multiple deoxyUridines are present without interfering with overall compound efficacywas used. In addition major improvement in tissue distribution andcellular uptake might be obtained by optimizing the structure of thehydrophobic conjugate. In some of the preferred embodiment the structureof sterol is modified to alter (increase/decrease) C17 attached chain.This type of modification results in significant increase in cellularuptake and improvement of tissue uptake prosperities in vivo.

dsRNA formulated according to the invention also includes rxRNAori.rxRNAori refers to a class of RNA molecules described in andincorporated by reference from PCT Publication No. WO2009/102427(Application No. PCT/US2009/000852), filed on Feb. 11, 2009, andentitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF.”

In some embodiments, an rxRNAori molecule comprises a double-strandedRNA (dsRNA) construct of 12-35 nucleotides in length, for inhibitingexpression of a target gene, comprising: a sense strand having a 5′-endand a 3′-end, wherein the sense strand is highly modified with21-modified ribose sugars, and wherein 3-6 nucleotides in the centralportion of the sense strand are not modified with 21-modified ribosesugars and, an antisense strand having a 5′-end and a 3′-end, whichhybridizes to the sense strand and to mRNA of the target gene, whereinthe dsRNA inhibits expression of the target gene in a sequence-dependentmanner.

rxRNAori can contain any of the modifications described herein. In someembodiments, at least 30% of the nucleotides in the rxRNAori aremodified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the rxRNAori aremodified. In some embodiments, 100% of the nucleotides in the sd-rxRNAare modified. In some embodiments, only the passenger strand of therxRNAori contains modifications.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Thus, aspects of the invention relate to isolated double strandednucleic acid molecules comprising a guide (antisense) strand and apassenger (sense) strand. As used herein, the term “double-stranded”refers to one or more nucleic acid molecules in which at least a portionof the nucleomonomers are complementary and hydrogen bond to form adouble-stranded region. In some embodiments, the length of the guidestrand ranges from 16-29 nucleotides long. In certain embodiments, theguide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or29 nucleotides long. The guide strand has complementarity to a targetgene. Complementarity between the guide strand and the target gene mayexist over any portion of the guide strand. Complementarity as usedherein may be perfect complementarity or less than perfectcomplementarity as long as the guide strand is sufficientlycomplementary to the target that it mediates RNAi. In some embodimentscomplementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%,or 1% mismatch between the guide strand and the target. Perfectcomplementarity refers to 100% complementarity. Thus the invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Moreover, not allpositions of a siRNA contribute equally to target recognition.Mismatches in the center of the siRNA are most critical and essentiallyabolish target RNA cleavage. Mismatches upstream of the center orupstream of the cleavage site referencing the antisense strand aretolerated but significantly reduce target RNA cleavage. Mismatchesdownstream of the center or cleavage site referencing the antisensestrand, preferably located near the 3′ end of the antisense strand, e.g.1, 2, 3, 4, 5 or 6 nucleotides from the 3′ end of the antisense strand,are tolerated and reduce target RNA cleavage only slightly.

While not wishing to be bound by any particular theory, in someembodiments, the guide strand is at least 16 nucleotides in length andanchors the Argonaute protein in RISC. In some embodiments, when theguide strand loads into RISC it has a defined seed region and targetmRNA cleavage takes place across from position 10-11 of the guidestrand. In some embodiments, the 5′ end of the guide strand is or isable to be phosphorylated. The nucleic acid molecules described hereinmay be referred to as minimum trigger RNA.

In some embodiments, the length of the passenger strand ranges from 8-15nucleotides long. In certain embodiments, the passenger strand is 8, 9,10, 11, 12, 13, 14 or 15 nucleotides long. The passenger strand hascomplementarity to the guide strand. Complementarity between thepassenger strand and the guide strand can exist over any portion of thepassenger or guide strand. In some embodiments, there is 100%complementarity between the guide and passenger strands within thedouble stranded region of the molecule.

Aspects of the invention relate to double stranded nucleic acidmolecules with minimal double stranded regions. In some embodiments theregion of the molecule that is double stranded ranges from 8-15nucleotides long. In certain embodiments, the region of the moleculethat is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotideslong. In certain embodiments the double stranded region is 13 or 14nucleotides long. There can be 100% complementarity between the guideand passenger strands, or there may be one or more mismatches betweenthe guide and passenger strands. In some embodiments, on one end of thedouble stranded molecule, the molecule is either blunt-ended or has aone-nucleotide overhang. The single stranded region of the molecule isin some embodiments between 4-12 nucleotides long. For example thesingle stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotideslong. However, in certain embodiments, the single stranded region canalso be less than 4 or greater than 12 nucleotides long. In certainembodiments, the single stranded region is at least 6 or at least 7nucleotides long.

RNAi constructs associated with the invention can have a thermodynamicstability (ΔG) of less than −13 kkal/mol. In some embodiments, thethermodynamic stability (ΔG) is less than −20 kkal/mol. In someembodiments there is a loss of efficacy when (ΔG) goes below −21kkal/mol. In some embodiments a (ΔG) value higher than −13 kkal/mol iscompatible with aspects of the invention. Without wishing to be bound byany theory, in some embodiments a molecule with a relatively higher (ΔG)value may become active at a relatively higher concentration, while amolecule with a relatively lower (ΔG) value may become active at arelatively lower concentration. In some embodiments, the (ΔG) value maybe higher than −9 kkcal/mol. The gene silencing effects mediated by theRNAi constructs associated with the invention, containing minimal doublestranded regions, are unexpected because molecules of almost identicaldesign but lower thermodynamic stability have been demonstrated to beinactive (Rana et al 2004).

Without wishing to be bound by any theory, results described hereinsuggest that a stretch of 8-10 bp of dsRNA or dsDNA will be structurallyrecognized by protein components of RISC or co-factors of RISC.Additionally, there is a free energy requirement for the triggeringcompound that it may be either sensed by the protein components and/orstable enough to interact with such components so that it may be loadedinto the Argonaute protein. If optimal thermodynamics are present andthere is a double stranded portion that is preferably at least 8nucleotides then the duplex will be recognized and loaded into the RNAimachinery.

In some embodiments, thermodynamic stability is increased through theuse of LNA bases. In some embodiments, additional chemical modificationsare introduced. Several non-limiting examples of chemical modificationsinclude: 5′ Phosphate, 2′-O-methyl, 2′-β-ethyl, 2′-fluoro,ribothymidine, C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU); C-5propynyl-C (pC) and C-5 propynyl-U (pU); 5-methyl C, 5-methyl U,5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine and MGB (minor groovebinder). It should be appreciated that more than one chemicalmodification can be combined within the same molecule.

Molecules associated with the invention are optimized for increasedpotency and/or reduced toxicity. For example, nucleotide length of theguide and/or passenger strand, and/or the number of phosphorothioatemodifications in the guide and/or passenger strand, can in some aspectsinfluence potency of the RNA molecule, while replacing 2′-fluoro (2′F)modifications with 2′-O-methyl (2′OMe) modifications can in some aspectsinfluence toxicity of the molecule. Specifically, reduction in 2′Fcontent of a molecule is predicted to reduce toxicity of the molecule.The Examples section presents molecules in which 2′F modifications havebeen eliminated, offering an advantage over previously described RNAicompounds due to a predicted reduction in toxicity. Furthermore, thenumber of phosphorothioate modifications in an RNA molecule caninfluence the uptake of the molecule into a cell, for example theefficiency of passive uptake of the molecule into a cell. Preferredembodiments of molecules described herein have no 2′F modification andyet are characterized by equal efficacy in cellular uptake and tissuepenetration. Such molecules represent a significant improvement overprior art, such as molecules described by Accell and Wolfrum, which areheavily modified with extensive use of 2′F.

In some embodiments, a guide strand is approximately 18-19 nucleotidesin length and has approximately 2-14 phosphate modifications. Forexample, a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more than 14 nucleotides that are phosphate-modified. Theguide strand may contain one or more modifications that confer increasedstability without interfering with RISC entry. The phosphate modifiednucleotides, such as phosphorothioate modified nucleotides, can be atthe 3′ end, 5′ end or spread throughout the guide strand. In someembodiments, the 3′ terminal 10 nucleotides of the guide strand contains1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides.The guide strand can also contain 2′F and/or 2′OMe modifications, whichcan be located throughout the molecule. In some embodiments, thenucleotide in position one of the guide strand (the nucleotide in themost 5′ position of the guide strand) is 2′OMe modified and/orphosphorylated. C and U nucleotides within the guide strand can be 2′Fmodified. For example, C and U nucleotides in positions 2-10 of a 19 ntguide strand (or corresponding positions in a guide strand of adifferent length) can be 2′F modified. C and U nucleotides within theguide strand can also be 2′OMe modified. For example, C and Unucleotides in positions 11-18 of a 19 nt guide strand (or correspondingpositions in a guide strand of a different length) can be 2′OMemodified. In some embodiments, the nucleotide at the most 3′ end of theguide strand is unmodified. In certain embodiments, the majority of Csand Us within the guide strand are 2′F modified and the 5′ end of theguide strand is phosphorylated. In other embodiments, position 1 and theCs or Us in positions 11-18 are 2′OMe modified and the 5′ end of theguide strand is phosphorylated. In other embodiments, position 1 and theCs or Us in positions 11-18 are 2′ OMe modified, the 5′ end of the guidestrand is phosphorylated, and the Cs or Us in position 2-10 are 2′Fmodified.

In some aspects, an optimal passenger strand is approximately 11-14nucleotides in length. The passenger strand may contain modificationsthat confer increased stability. One or more nucleotides in thepassenger strand can be 2′ OMe modified. In some embodiments, one ormore of the C and/or U nucleotides in the passenger strand is 2′ OMemodified, or all of the C and U nucleotides in the passenger strand are2′OMe modified. In certain embodiments, all of the nucleotides in thepassenger strand are 2′ OMe modified. One or more of the nucleotides onthe passenger strand can also be phosphate-modified such asphosphorothioate modified. The passenger strand can also contain 2′ribo, 2′F and 2 deoxy modifications or any combination of the above. Asdemonstrated in the Examples, chemical modification patterns on both theguide and passenger strand are well tolerated and a combination ofchemical modifications is shown herein to lead to increased efficacy andself-delivery of RNA molecules.

Aspects of the invention relate to RNAi constructs that have extendedsingle-stranded regions relative to double stranded regions, as comparedto molecules that have been used previously for RNAi. The singlestranded region of the molecules may be modified to promote cellularuptake or gene silencing. In some embodiments, phosphorothioatemodification of the single stranded region influences cellular uptakeand/or gene silencing. The region of the guide strand that isphosphorothioate modified can include nucleotides within both the singlestranded and double stranded regions of the molecule. In someembodiments, the single stranded region includes 2-12 phosphorothioatemodifications. For example, the single stranded region can include 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In someinstances, the single stranded region contains 6-8 phosphorothioatemodifications.

Molecules associated with the invention are also optimized for cellularuptake. In RNA molecules described herein, the guide and/or passengerstrands can be attached to a conjugate. In certain embodiments theconjugate is hydrophobic. The hydrophobic conjugate can be a smallmolecule with a partition coefficient that is higher than 10. Theconjugate can be a sterol-type molecule such as cholesterol, or amolecule with an increased length polycarbon chain attached to C17, andthe presence of a conjugate can influence the ability of an RNA moleculeto be taken into a cell with or without a lipid transfection reagent.The conjugate can be attached to the passenger or guide strand through ahydrophobic linker In some embodiments, a hydrophobic linker is 5-12C inlength, and/or is hydroxypyrrolidine-based. In some embodiments, ahydrophobic conjugate is attached to the passenger strand and the CUresidues of either the passenger and/or guide strand are modified. Insome embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or 95% of the CU residues on the passenger strand and/or the guidestrand are modified. In some aspects, molecules associated with theinvention are self-delivering (sd). As used herein, “self-delivery”refers to the ability of a molecule to be delivered into a cell withoutthe need for an additional delivery vehicle such as a transfectionreagent.

Aspects of the invention relate to selecting molecules for use in RNAi.In some embodiments, molecules that have a double stranded region of8-15 nucleotides can be selected for use in RNAi. In some embodiments,molecules are selected based on their thermodynamic stability (ΔG). Insome embodiments, molecules will be selected that have a (ΔG) of lessthan −13 kkal/mol. For example, the (ΔG) value may be −13, −14, −15,−16, −17, −18, −19, −21, −22 or less than −22 kkal/mol. In otherembodiments, the (ΔG) value may be higher than −13 kkal/mol. Forexample, the (ΔG) value may be −12, −11, −10, −9, −8, −7 or more than −7kkal/mol. It should be appreciated that AG can be calculated using anymethod known in the art. In some embodiments AG is calculated usingMfold, available through the Mfold internet site(mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi). Methods for calculatingAG are described in, and are incorporated by reference from, thefollowing references: Zuker, M. (2003) Nucleic Acids Res.,31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H.(1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs,J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl.Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H.(2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I.L., and Schuster, P. (1999) Biopolymers 49:145-165.

In certain embodiments, the polynucleotide contains 5′- and/or 3′-endoverhangs. The number and/or sequence of nucleotides overhang on one endof the polynucleotide may be the same or different from the other end ofthe polynucleotide. In certain embodiments, one or more of the overhangnucleotides may contain chemical modification(s), such asphosphorothioate or 2′-OMe modification.

In certain embodiments, the polynucleotide is unmodified. In otherembodiments, at least one nucleotide is modified. In furtherembodiments, the modification includes a 2′-H or 2′-modified ribosesugar at the 2nd nucleotide from the 5′-end of the guide sequence. The“2nd nucleotide” is defined as the second nucleotide from the 5′-end ofthe polynucleotide.

As used herein, “2′-modified ribose sugar” includes those ribose sugarsthat do not have a 2′-OH group. “2′-modified ribose sugar” does notinclude 2′-deoxyribose (found in unmodified canonical DNA nucleotides).For example, the 2′-modified ribose sugar may be 2′-O-alkyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, or combinationthereof.

In certain embodiments, the 2′-modified nucleotides are pyrimidinenucleotides (e.g., C/U). Examples of 2′-O-alkyl nucleotides include2′-O-methyl nucleotides, or 2′-O-allyl nucleotides.

In certain embodiments, the sd-rxRNA polynucleotide of the inventionwith the above-referenced 5′-end modification exhibits significantly(e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or more) less “off-target” gene silencing whencompared to similar constructs without the specified 5′-endmodification, thus greatly improving the overall specificity of the RNAireagent or therapeutics.

As used herein, “off-target” gene silencing refers to unintended genesilencing due to, for example, spurious sequence homology between theantisense (guide) sequence and the unintended target mRNA sequence.

According to this aspect of the invention, certain guide strandmodifications further increase nuclease stability, and/or lowerinterferon induction, without significantly decreasing RNAi activity (orno decrease in RNAi activity at all).

In some embodiments, the 5′-stem sequence may comprise a 2′-modifiedribose sugar, such as 2′-O-methyl modified nucleotide, at the 2^(nd)nucleotide on the 5′-end of the polynucleotide and, in some embodiments,no other modified nucleotides. The hairpin structure having suchmodification may have enhanced target specificity or reduced off-targetsilencing compared to a similar construct without the 2′-O-methylmodification at said position.

Certain combinations of specific 5′-stem sequence and 3′-stem sequencemodifications may result in further unexpected advantages, as partlymanifested by enhanced ability to inhibit target gene expression,enhanced serum stability, and/or increased target specificity, etc.

In certain embodiments, the guide strand comprises a 2′-O-methylmodified nucleotide at the 2^(nd) nucleotide on the 5′-end of the guidestrand and no other modified nucleotides.

In other aspects, the sd-rxRNA structures of the present inventionmediates sequence-dependent gene silencing by a microRNA mechanism. Asused herein, the term “microRNA” (“miRNA”), also referred to in the artas “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g., by viral,mammalian, or plant genomes) and are capable of directing or mediatingRNA silencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

microRNAs are involved in down-regulating target genes in criticalpathways, such as development and cancer, in mice, worms and mammals.Gene silencing through a microRNA mechanism is achieved by specific yetimperfect base-pairing of the miRNA and its target messenger RNA (mRNA).Various mechanisms may be used in microRNA-mediated down-regulation oftarget mRNA expression.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAs areexpressed by endogenous genes in vivo and are processed from a hairpinor stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or otherRNAses. miRNAs can exist transiently in vivo as a double-stranded duplexbut only one strand is taken up by the RISC complex to direct genesilencing.

In some embodiments a version of sd-rxRNA compounds, which are effectivein cellular uptake and inhibiting of miRNA activity are described.Essentially the compounds are similar to RISC entering version but largestrand chemical modification patterns are optimized in the way to blockcleavage and act as an effective inhibitor of the RISC action. Forexample, the compound might be completely or mostly Omethyl modifiedwith the PS content described previously. For these types of compoundsthe 5′ phosphorylation is not necessary. The presence of double strandedregion is preferred as it is promotes cellular uptake and efficient RISCloading.

Another pathway that uses small RNAs as sequence-specific regulators isthe RNA interference (RNAi) pathway, which is an evolutionarilyconserved response to the presence of double-stranded RNA (dsRNA) in thecell. The dsRNAs are cleaved into ˜20-base pair (bp) duplexes ofsmall-interfering RNAs (siRNAs) by Dicer. These small RNAs get assembledinto multiprotein effector complexes called RNA-induced silencingcomplexes (RISCs). The siRNAs then guide the cleavage of target mRNAswith perfect complementarity.

Some aspects of biogenesis, protein complexes, and function are sharedbetween the siRNA pathway and the miRNA pathway. The subjectsingle-stranded polynucleotides may mimic the dsRNA in the siRNAmechanism, or the microRNA in the miRNA mechanism.

In certain embodiments, the modified RNAi constructs may have improvedstability in serum and/or cerebral spinal fluid compared to anunmodified RNAi constructs having the same sequence.

In certain embodiments, the structure of the RNAi construct does notinduce interferon response in primary cells, such as mammalian primarycells, including primary cells from human, mouse and other rodents, andother non-human mammals. In certain embodiments, the RNAi construct mayalso be used to inhibit expression of a target gene in an invertebrateorganism.

To further increase the stability of the subject constructs in vivo, the3′-end of the hairpin structure may be blocked by protective group(s).For example, protective groups such as inverted nucleotides, invertedabasic moieties, or amino-end modified nucleotides may be used. Invertednucleotides may comprise an inverted deoxynucleotide. Inverted abasicmoieties may comprise an inverted deoxyabasic moiety, such as a3′,3′-linked or 5′,5′-linked deoxyabasic moiety.

The RNAi constructs of the invention are capable of inhibiting thesynthesis of any target protein encoded by target gene(s). The inventionincludes methods to inhibit expression of a target gene either in a cellin vitro, or in vivo. As such, the RNAi constructs of the invention areuseful for treating a patient with a disease characterized by theoverexpression of a target gene.

The target gene can be endogenous or exogenous (e.g., introduced into acell by a virus or using recombinant DNA technology) to a cell. Suchmethods may include introduction of RNA into a cell in an amountsufficient to inhibit expression of the target gene. By way of example,such an RNA molecule may have a guide strand that is complementary tothe nucleotide sequence of the target gene, such that the compositioninhibits expression of the target gene.

The invention also relates to vectors expressing the nucleic acids ofthe invention, and cells comprising such vectors or the nucleic acids.The cell may be a mammalian cell in vivo or in culture, such as a humancell.

The invention further relates to compositions comprising the subjectRNAi constructs, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention provides a method for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingan eye cell with any of the subject RNAi constructs.

The method may be carried out in vitro, ex vivo, or in vivo, in, forexample, mammalian cells in culture, such as a human cell in culture.

The target cells (e.g., mammalian cell) may be contacted in the presenceof a delivery reagent, such as a lipid (e.g., a cationic lipid) or aliposome.

Another aspect of the invention provides a method for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingthe mammalian cell with a vector expressing the subject RNAi constructs.

In one aspect of the invention, a longer duplex polynucleotide isprovided, including a first polynucleotide that ranges in size fromabout 16 to about 30 nucleotides; a second polynucleotide that ranges insize from about 26 to about 46 nucleotides, wherein the firstpolynucleotide (the antisense strand) is complementary to both thesecond polynucleotide (the sense strand) and a target gene, and whereinboth polynucleotides form a duplex and wherein the first polynucleotidecontains a single stranded region longer than 6 bases in length and ismodified with alternative chemical modification pattern, and/or includesa conjugate moiety that facilitates cellular delivery. In thisembodiment, between about 40% to about 90% of the nucleotides of thepassenger strand between about 40% to about 90% of the nucleotides ofthe guide strand, and between about 40% to about 90% of the nucleotidesof the single stranded region of the first polynucleotide are chemicallymodified nucleotides.

In an embodiment, the chemically modified nucleotide in thepolynucleotide duplex may be any chemically modified nucleotide known inthe art, such as those discussed in detail above. In a particularembodiment, the chemically modified nucleotide is selected from thegroup consisting of 2′ F modified nucleotides, 2′-O-methyl modified and2′ deoxy nucleotides. In another particular embodiment, the chemicallymodified nucleotides results from “hydrophobic modifications” of thenucleotide base. In another particular embodiment, the chemicallymodified nucleotides are phosphorothioates. In an additional particularembodiment, chemically modified nucleotides are combination ofphosphorothioates, 2′-O-methyl, 2′ deoxy, hydrophobic modifications andphosphorothioates. As these groups of modifications refer tomodification of the ribose ring, back bone and nucleotide, it isfeasible that some modified nucleotides will carry a combination of allthree modification types.

In another embodiment, the chemical modification is not the same acrossthe various regions of the duplex. In a particular embodiment, the firstpolynucleotide (the passenger strand), has a large number of diversechemical modifications in various positions. For this polynucleotide upto 90% of nucleotides might be chemically modified and/or havemismatches introduced.

In another embodiment, chemical modifications of the first or secondpolynucleotide include, but not limited to, 5′ position modification ofUridine and Cytosine (4-pyridyl, 2-pyridyl, indolyl, phenyl (C₆H₅OH);tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl;naphthyl, etc), where the chemical modification might alter base pairingcapabilities of a nucleotide. For the guide strand an important featureof this aspect of the invention is the position of the chemicalmodification relative to the 5′ end of the antisense and sequence. Forexample, chemical phosphorylation of the 5′ end of the guide strand isusually beneficial for efficacy. O-methyl modifications in the seedregion of the sense strand (position 2-7 relative to the 5′ end) are notgenerally well tolerated, whereas 2′F and deoxy are well tolerated. Themid part of the guide strand and the 3′ end of the guide strand are morepermissive in a type of chemical modifications applied. Deoxymodifications are not tolerated at the 3′ end of the guide strand.

A unique feature of this aspect of the invention involves the use ofhydrophobic modification on the bases. In one embodiment, thehydrophobic modifications are preferably positioned near the 5′ end ofthe guide strand, in other embodiments, they localized in the middle ofthe guides strand, in other embodiment they localized at the 3′ end ofthe guide strand and yet in another embodiment they are distributedthought the whole length of the polynucleotide. The same type ofpatterns is applicable to the passenger strand of the duplex.

The other part of the molecule is a single stranded region. The singlestranded region is expected to range from 7 to 40 nucleotides.

In one embodiment, the single stranded region of the firstpolynucleotide contains modifications selected from the group consistingof between 40% and 90% hydrophobic base modifications, between 40%-90%phosphorothioates, between 40%-90% modification of the ribose moiety,and any combination of the preceding.

Efficiency of guide strand (first polynucleotide) loading into the RISCcomplex might be altered for heavily modified polynucleotides, so in oneembodiment, the duplex polynucleotide includes a mismatch betweennucleotide 9, 11, 12, 13, or 14 on the guide strand (firstpolynucleotide) and the opposite nucleotide on the sense strand (secondpolynucleotide) to promote efficient guide strand loading.

More detailed aspects of the invention are described in the sectionsbelow.

Duplex Characteristics

Double-stranded oligonucleotides of the invention may be formed by twoseparate complementary nucleic acid strands. Duplex formation can occureither inside or outside the cell containing the target gene.

As used herein, the term “duplex” includes the region of thedouble-stranded nucleic acid molecule(s) that is (are) hydrogen bondedto a complementary sequence. Double-stranded oligonucleotides of theinvention may comprise a nucleotide sequence that is sense to a targetgene and a complementary sequence that is antisense to the target gene.The sense and antisense nucleotide sequences correspond to the targetgene sequence, e.g., are identical or are sufficiently identical toeffect target gene inhibition (e.g., are about at least about 98%identical, 96% identical, 94%, 90% identical, 85% identical, or 80%identical) to the target gene sequence.

In certain embodiments, the double-stranded oligonucleotide of theinvention is double-stranded over its entire length, i.e., with nooverhanging single-stranded sequence at either end of the molecule,i.e., is blunt-ended. In other embodiments, the individual nucleic acidmolecules can be of different lengths. In other words, a double-strandedoligonucleotide of the invention is not double-stranded over its entirelength. For instance, when two separate nucleic acid molecules are used,one of the molecules, e.g., the first molecule comprising an antisensesequence, can be longer than the second molecule hybridizing thereto(leaving a portion of the molecule single-stranded). Likewise, when asingle nucleic acid molecule is used a portion of the molecule at eitherend can remain single-stranded.

In one embodiment, a double-stranded oligonucleotide of the inventioncontains mismatches and/or loops or bulges, but is double-stranded overat least about 70% of the length of the oligonucleotide. In anotherembodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over at least about 80% of the length of theoligonucleotide. In another embodiment, a double-strandedoligonucleotide of the invention is double-stranded over at least about90%-95% of the length of the oligonucleotide. In another embodiment, adouble-stranded oligonucleotide of the invention is double-stranded overat least about 96%-98% of the length of the oligonucleotide. In certainembodiments, the double-stranded oligonucleotide of the inventioncontains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 mismatches.

Modifications

The nucleotides of the invention may be modified at various locations,including the sugar moiety, the phosphodiester linkage, and/or the base.

In some embodiments, the base moiety of a nucleoside may be modified.For example, a pyrimidine base may be modified at the 2, 3, 4, 5, and/or6 position of the pyrimidine ring. In some embodiments, the exocyclicamine of cytosine may be modified. A purine base may also be modified.For example, a purine base may be modified at the 1, 2, 3, 6, 7, or 8position. In some embodiments, the exocyclic amine of adenine may bemodified. In some cases, a nitrogen atom in a ring of a base moiety maybe substituted with another atom, such as carbon. A modification to abase moiety may be any suitable modification. Examples of modificationsare known to those of ordinary skill in the art. In some embodiments,the base modifications include alkylated purines or pyrimidines,acylated purines or pyrimidines, or other heterocycles.

In some embodiments, a pyrimidine may be modified at the 5 position. Forexample, the 5 position of a pyrimidine may be modified with an alkylgroup, an alkynyl group, an alkenyl group, an acyl group, or substitutedderivatives thereof. In other examples, the 5 position of a pyrimidinemay be modified with a hydroxyl group or an alkoxyl group or substitutedderivative thereof. Also, the N⁴ position of a pyrimidine may bealkylated. In still further examples, the pyrimidine 5-6 bond may besaturated, a nitrogen atom within the pyrimidine ring may be substitutedwith a carbon atom, and/or the O² and O⁴ atoms may be substituted withsulfur atoms. It should be understood that other modifications arepossible as well.

In other examples, the N⁷ position and/or N² and/or N³ position of apurine may be modified with an alkyl group or substituted derivativethereof. In further examples, a third ring may be fused to the purinebicyclic ring system and/or a nitrogen atom within the purine ringsystem may be substituted with a carbon atom. It should be understoodthat other modifications are possible as well.

Non-limiting examples of pyrimidines modified at the 5 position aredisclosed in U.S. Pat. No. 5,591,843, U.S. Pat. No. 7,205,297, U.S. Pat.No. 6,432,963, and U.S. Pat. No. 6,020,483; non-limiting examples ofpyrimidines modified at the N⁴ position are disclosed in U.S. Pat. No.5,580,731; non-limiting examples of purines modified at the 8 positionare disclosed in U.S. Pat. No. 6,355,787 and U.S. Pat. No. 5,580,972;non-limiting examples of purines modified at the N⁶ position aredisclosed in U.S. Pat. No. 4,853,386, U.S. Pat. No. 5,789,416, and U.S.Pat. No. 7,041,824; and non-limiting examples of purines modified at the2 position are disclosed in U.S. Pat. No. 4,201,860 and U.S. Pat. No.5,587,469, all of which are incorporated herein by reference.

Non-limiting examples of modified bases include N⁴,N⁴-ethanocytosine,7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N⁶-methyladenine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyl uracil, dihydrouracil, inosine,N⁶-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine, and2,6-diaminopurine. In some embodiments, the base moiety may be aheterocyclic base other than a purine or pyrimidine. The heterocyclicbase may be optionally modified and/or substituted.

Sugar moieties include natural, unmodified sugars, e.g., monosaccharide(such as pentose, e.g., ribose, deoxyribose), modified sugars and sugaranalogs. In general, possible modifications of nucleomonomers,particularly of a sugar moiety, include, for example, replacement of oneor more of the hydroxyl groups with a halogen, a heteroatom, analiphatic group, or the functionalization of the hydroxyl group as anether, an amine, a thiol, or the like.

One particularly useful group of modified nucleomonomers are 2′-O-methylnucleotides. Such 2′-O-methyl nucleotides may be referred to as“methylated,” and the corresponding nucleotides may be made fromunmethylated nucleotides followed by alkylation or directly frommethylated nucleotide reagents. Modified nucleomonomers may be used incombination with unmodified nucleomonomers. For example, anoligonucleotide of the invention may contain both methylated andunmethylated nucleomonomers.

Some exemplary modified nucleomonomers include sugar- orbackbone-modified ribonucleotides. Modified ribonucleotides may containa non-naturally occurring base (instead of a naturally occurring base),such as uridines or cytidines modified at the 5′-position, e.g.,5′-(2-amino)propyl uridine and 5′-bromo uridine; adenosines andguanosines modified at the 8-position, e.g., 8-bromo guanosine; deazanucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g.,N⁶-methyl adenosine. Also, sugar-modified ribonucleotides may have the2′-OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH,SR, amino (such as NH₂, NHR, NR₂,), or CN group, wherein R is loweralkyl, alkenyl, or alkynyl.

Modified ribonucleotides may also have the phosphodiester groupconnecting to adjacent ribonucleotides replaced by a modified group,e.g., of phosphorothioate group. More generally, the various nucleotidemodifications may be combined.

Although the antisense (guide) strand may be substantially identical toat least a portion of the target gene (or genes), at least with respectto the base pairing properties, the sequence need not be perfectlyidentical to be useful, e.g., to inhibit expression of a target gene'sphenotype. Generally, higher homology can be used to compensate for theuse of a shorter antisense gene. In some cases, the antisense strandgenerally will be substantially identical (although in antisenseorientation) to the target gene.

The use of 2′-O-methyl modified RNA may also be beneficial incircumstances in which it is desirable to minimize cellular stressresponses. RNA having 2′-O-methyl nucleomonomers may not be recognizedby cellular machinery that is thought to recognize unmodified RNA. Theuse of 2′-O-methylated or partially 2′-O-methylated RNA may avoid theinterferon response to double-stranded nucleic acids, while maintainingtarget RNA inhibition. This may be useful, for example, for avoiding theinterferon or other cellular stress responses, both in short RNAi (e.g.,siRNA) sequences that induce the interferon response, and in longer RNAisequences that may induce the interferon response.

Overall, modified sugars may include D-ribose, 2′-O-alkyl (including2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl,2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy(—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, andcyano and the like. In one embodiment, the sugar moiety can be a hexoseand incorporated into an oligonucleotide as described (Augustyns, K., etal., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can befound, e.g., in U.S. Pat. No. 5,849,902, incorporated by referenceherein.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

In certain embodiments, oligonucleotides of the invention comprise 3′and 5′ termini (except for circular oligonucleotides). In oneembodiment, the 3′ and 5′ termini of an oligonucleotide can besubstantially protected from nucleases e.g., by modifying the 3′ or 5′linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example,oligonucleotides can be made resistant by the inclusion of a “blockinggroup.” The term “blocking group” as used herein refers to substituents(e.g., other than OH groups) that can be attached to oligonucleotides ornucleomonomers, either as protecting groups or coupling groups forsynthesis (e.g., FITC, propyl (CH₂—CH₂—CH₃), glycol (—O—CH₂—CH₂—O—)phosphate (PO₃ ²), hydrogen phosphonate, or phosphoramidite). “Blockinggroups” also include “end blocking groups” or “exonuclease blockinggroups” which protect the 5′ and 3′ termini of the oligonucleotide,including modified nucleotides and non-nucleotide exonuclease resistantstructures.

Exemplary end-blocking groups include cap structures (e.g., a7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or5′-5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res.Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups(e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The3′ terminal nucleomonomer comprises a 3′-O that can optionally besubstituted by a blocking group that prevents 3′-exonuclease degradationof the oligonucleotide. For example, the 3′-hydroxyl can be esterifiedto a nucleotide through a 3′→3′ internucleotide linkage. For example,the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, andpreferably, ethoxy. Optionally, the 3′→3′linked nucleotide at the 3′terminus can be linked by a substitute linkage. To reduce nucleasedegradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g.,a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably,the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′terminal hydroxy moiety can be esterified with a phosphorus containingmoiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In certain embodiments, a protectinggroup reacts selectively in good yield to give a protected substratethat is stable to the projected reactions; the protecting group shouldbe selectively removable in good yield by readily available, preferablynon-toxic reagents that do not attack the other functional groups; theprotecting group forms an easily separable derivative (more preferablywithout the generation of new stereogenic centers); and the protectinggroup has a minimum of additional functionality to avoid further sitesof reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbonprotecting groups may be utilized. Hydroxyl protecting groups includemethyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Amino-protecting groups include methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Exemplary protecting groups are detailed herein. However, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described in Protective Groups in OrganicSynthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceeded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. Heteroatoms such as nitrogen may have hydrogen substituentsand/or any permissible substituents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Furthermore, thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds. Combinations of substituents andvariables envisioned by this invention are preferably those that resultin the formation of stable compounds useful in the treatment, forexample, of infectious diseases or proliferative disorders. The term“stable”, as used herein, preferably refers to compounds which possessstability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, acyclic,cyclic, or polycyclic aliphatic hydrocarbons, which are optionallysubstituted with one or more functional groups. As will be appreciatedby one of ordinary skill in the art, “aliphatic” is intended herein toinclude, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term“alkyl” includes straight, branched and cyclic alkyl groups. Ananalogous convention applies to other generic terms such as “alkenyl,”“alkynyl,” and the like. Furthermore, as used herein, the terms “alkyl,”“alkenyl,” “alkynyl,” and the like encompass both substituted andunsubstituted groups. In certain embodiments, as used herein, “loweralkyl” is used to indicate those alkyl groups (cyclic, acyclic,substituted, unsubstituted, branched, or unbranched) having 1-6 carbonatoms.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,—CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments described herein.

The term “heteroaliphatic,” as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has 6or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms.

Moreover, unless otherwise specified, the term alkyl includes both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “alkylaryl” or an “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond. For example, the term “alkenyl”includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.),branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups(cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certainembodiments, a straight chain or branched chain alkenyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groupscontaining 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkenyl includes both“unsubstituted alkenyls” and “substituted alkenyls,” the latter of whichrefers to alkenyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond. For example, the term “alkynyl”includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.),branched-chain alkynyl groups, and cycloalkyl or cycloalkenylsubstituted alkynyl groups. In certain embodiments, a straight chain orbranched chain alkynyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The termC₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkynyl includes both“unsubstituted alkynyls” and “substituted alkynyls,” the latter of whichrefers to alkynyl moieties having independently selected substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, alkyl groups, alkynylgroups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withindependently selected groups such as alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffiydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.Examples of halogen substituted alkoxy groups include, but are notlimited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻(with an appropriate counterion).

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The term “substituted” includes independently selected substituentswhich can be placed on the moiety and which allow the molecule toperform its intended function. Examples of substituents include alkyl,alkenyl, alkynyl, aryl, (CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃CN, NO₂, halogen,(CR′R″)₀₋₃C(halogen)₃, (CR′R″)₀₋₃CH(halogen)₂, (CR′R″)₀₋₃CH₂(halogen),(CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO,(CRR″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃S(O)₀₋₂R′, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃COR′, (CR′R″)₀₋₃CO₂R′, or (CR′R″)₀₋₃OR′ groups; wherein eachR′ and R″ are each independently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl,C₂-C₅ alkynyl, or aryl group, or R′ and R″ taken together are abenzylidene group or a —(CH₂)₂—O—(CH₂)₂— group.

The term “amine” or “amino” includes compounds or moieties in which anitrogen atom is covalently bonded to at least one carbon or heteroatom.The term “alkyl amino” includes groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,”“nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide”refer to a polymer of two or more nucleotides. The polynucleotides canbe DNA, RNA, or derivatives or modified versions thereof. Thepolynucleotide may be single-stranded or double-stranded. Thepolynucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,its hybridization parameters, etc. The polynucleotide may comprise amodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Theolynucleotide may comprise a modified sugar moiety (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose,and hexose), and/or a modified phosphate moiety (e.g., phosphorothioatesand 5′-N-phosphoramidite linkages). A nucleotide sequence typicallycarries genetic information, including the information used by cellularmachinery to make proteins and enzymes. These terms include double- orsingle-stranded genomic and cDNA, RNA, any synthetic and geneticallymanipulated polynucleotide, and both sense and antisensepolynucleotides. This includes single- and double-stranded molecules,i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleicacids” (PNA) formed by conjugating bases to an amino acid backbone.

The term “base” includes the known purine and pyrimidine heterocyclicbases, deazapurines, and analogs (including heterocyclic substitutedanalogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-,1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomersthereof. Examples of purines include adenine, guanine, inosine,diaminopurine, and xanthine and analogs (e.g., 8-oxo-N⁶-methyladenine or7-diazaxanthine) and derivatives thereof. Pyrimidines include, forexample, thymine, uracil, and cytosine, and their analogs (e.g.,5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil,5-(1-propynyl)cytosine and 4,4-ethanocytosine). Other examples ofsuitable bases include non-purinyl and non-pyrimidinyl bases such as2-aminopyridine and triazines.

In a preferred embodiment, the nucleomonomers of an oligonucleotide ofthe invention are RNA nucleotides. In another preferred embodiment, thenucleomonomers of an oligonucleotide of the invention are modified RNAnucleotides. Thus, the oligunucleotides contain modified RNAnucleotides.

The term “nucleoside” includes bases which are covalently attached to asugar moiety, preferably ribose or deoxyribose. Examples of preferrednucleosides include ribonucleosides and deoxyribonucleosides.Nucleosides also include bases linked to amino acids or amino acidanalogs which may comprise free carboxyl groups, free amino groups, orprotecting groups. Suitable protecting groups are well known in the art(see P. G. M. Wuts and T. W. Greene, “Protective Groups in OrganicSynthesis”, 2^(nd) Ed., Wiley-Interscience, New York, 1999).

The term “nucleotide” includes nucleosides which further comprise aphosphate group or a phosphate analog.

The nucleic acid molecules may be associated with a hydrophobic moietyfor targeting and/or delivery of the molecule to a cell. In certainembodiments, the hydrophobic moiety is associated with the nucleic acidmolecule through a linker. In certain embodiments, the association isthrough non-covalent interactions. In other embodiments, the associationis through a covalent bond. Any linker known in the art may be used toassociate the nucleic acid with the hydrophobic moiety. Linkers known inthe art are described in published international PCT applications, WO92/03464, WO 95/23162, WO 2008/021157, WO 2009/021157, WO 2009/134487,WO 2009/126933, U.S. Patent Application Publication 2005/0107325, U.S.Pat. No. 5,414,077, U.S. Pat. No. 5,419,966, U.S. Pat. No. 5,512,667,U.S. Pat. No. 5,646,126, and U.S. Pat. No. 5,652,359, which areincorporated herein by reference. The linker may be as simple as acovalent bond to a multi-atom linker. The linker may be cyclic oracyclic. The linker may be optionally substituted. In certainembodiments, the linker is capable of being cleaved from the nucleicacid. In certain embodiments, the linker is capable of being hydrolyzedunder physiological conditions. In certain embodiments, the linker iscapable of being cleaved by an enzyme (e.g., an esterase orphosphodiesterase). In certain embodiments, the linker comprises aspacer element to separate the nucleic acid from the hydrophobic moiety.The spacer element may include one to thirty carbon or heteroatoms. Incertain embodiments, the linker and/or spacer element comprisesprotonatable functional groups. Such protonatable functional groups maypromote the endosomal escape of the nucleic acid molecule. Theprotonatable functional groups may also aid in the delivery of thenucleic acid to a cell, for example, neutralizing the overall charge ofthe molecule. In other embodiments, the linker and/or spacer element isbiologically inert (that is, it does not impart biological activity orfunction to the resulting nucleic acid molecule).

In certain embodiments, the nucleic acid molecule with a linker andhydrophobic moiety is of the formulae described herein. In certainembodiments, the nucleic acid molecule is of the formula:

wherein

X is N or CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;

R¹ is a hydrophobic moiety;

R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; and

R³ is a nucleic acid.

In certain embodiments, the molecule is of the formula:

In certain embodiments, the molecule is of the formula:

In certain embodiments, the molecule is of the formula:

In certain embodiments, the molecule is of the formula:

In certain embodiments, X is N. In certain embodiments, X is CH.

In certain embodiments, A is a bond. In certain embodiments, A issubstituted or unsubstituted, cyclic or acyclic, branched or unbranchedaliphatic. In certain embodiments, A is acyclic, substituted orunsubstituted, branched or unbranched aliphatic. In certain embodiments,A is acyclic, substituted, branched or unbranched aliphatic. In certainembodiments, A is acyclic, substituted, unbranched aliphatic. In certainembodiments, A is acyclic, substituted, unbranched alkyl. In certainembodiments, A is acyclic, substituted, unbranched C₁₋₂₀ alkyl. Incertain embodiments, A is acyclic, substituted, unbranched C₁₋₁₂ alkyl.In certain embodiments, A is acyclic, substituted, unbranched C₁₋₁₀alkyl. In certain embodiments, A is acyclic, substituted, unbranchedC₁₋₈ alkyl. In certain embodiments, A is acyclic, substituted,unbranched C₁₋₆ alkyl. In certain embodiments, A is substituted orunsubstituted, cyclic or acyclic, branched or unbranchedheteroaliphatic. In certain embodiments, A is acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic. In certainembodiments, A is acyclic, substituted, branched or unbranchedheteroaliphatic. In certain embodiments, A is acyclic, substituted,unbranched heteroaliphatic.

In certain embodiments, A is of the formula:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

wherein

each occurrence of R is independently the side chain of a natural orunnatural amino acid; and

n is an integer between 1 and 20, inclusive. In certain embodiments, Ais of the formula:

In certain embodiments, each occurrence of R is independently the sidechain of a natural amino acid. In certain embodiments, n is an integerbetween 1 and 15, inclusive. In certain embodiments, n is an integerbetween 1 and 10, inclusive. In certain embodiments, n is an integerbetween 1 and 5, inclusive.

In certain embodiments, A is of the formula:

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, A is of the formula:

In certain embodiments, n is an integer between 1 and 15, inclusive. Incertain embodiments, n is an integer between 1 and 10, inclusive. Incertain embodiments, n is an integer between 1 and 5, inclusive.

In certain embodiments, A is of the formula:

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, A is of the formula:

In certain embodiments, n is an integer between 1 and 15, inclusive. Incertain embodiments, n is an integer between 1 and 10, inclusive. Incertain embodiments, n is an integer between 1 and 5, inclusive.

In certain embodiments, the molecule is of the formula:

wherein X, R¹, R², and R³ are as defined herein; and

A′ is substituted or unsubstituted, cyclic or acyclic, branched orunbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic.

In certain embodiments, A′ is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

In certain embodiments, R¹ is a steroid. In certain embodiments, R¹ is acholesterol. In certain embodiments, R¹ is a lipophilic vitamin. Incertain embodiments, R¹ is a vitamin A.

In certain embodiments, R¹ is a vitamin E.

In certain embodiments, R¹ is of the formula:

wherein R^(A) is substituted or unsubstituted, cyclic or acyclic,branched or unbranched aliphatic; or substituted or unsubstituted,cyclic or acyclic, branched or unbranched heteroaliphatic.

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

wherein

X is Nor CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;R¹ is a hydrophobic moiety;R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; andR³ is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein

X is Nor CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;R¹ is a hydrophobic moiety;R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; andR³ is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein

X is Nor CH;

A is a bond; substituted or unsubstituted, cyclic or acyclic, branchedor unbranched aliphatic; or substituted or unsubstituted, cyclic oracyclic, branched or unbranched heteroaliphatic;R¹ is a hydrophobic moiety;R² is hydrogen; an oxygen-protecting group; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; andR³ is a nucleic acid. In certain embodiments, the nucleic acid moleculeis of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

wherein R³ is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein R³ is a nucleic acid; andn is an integer between 1 and 20, inclusive.

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

As used herein, the term “linkage” includes a naturally occurring,unmodified phosphodiester moiety (−O—(PO²⁻)—O—) that covalently couplesadjacent nucleomonomers. As used herein, the term “substitute linkage”includes any analog or derivative of the native phosphodiester groupthat covalently couples adjacent nucleomonomers. Substitute linkagesinclude phosphodiester analogs, e.g., phosphorothioate,phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester,P-alkyloxyphosphotriester, methylphosphonate, and nonphosphoruscontaining linkages, e.g., acetals and amides. Such substitute linkagesare known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res.19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). Incertain embodiments, non-hydrolizable linkages are preferred, such asphosphorothiate linkages.

In certain embodiments, oligonucleotides of the invention comprisehydrophobicly modified nucleotides or “hydrophobic modifications.” Asused herein “hydrophobic modifications” refers to bases that aremodified such that (1) overall hydrophobicity of the base issignificantly increased, and/or (2) the base is still capable of formingclose to regular Watson—Crick interaction. Several non-limiting examplesof base modifications include 5-position uridine and cytidinemodifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, andisobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl,butyl, aminobenzyl; phenyl; and naphthyl.

Another type of conjugates that can be attached to the end (3′ or 5′end), the loop region, or any other parts of the sd-rxRNA might includea sterol, sterol type molecule, peptide, small molecule, protein, etc.In some embodiments, a sdrxRNA may contain more than one conjugates(same or different chemical nature). In some embodiments, the conjugateis cholesterol.

Another way to increase target gene specificity, or to reduce off-targetsilencing effect, is to introduce a 2′-modification (such as the 2′-Omethyl modification) at a position corresponding to the second 5′-endnucleotide of the guide sequence. This allows the positioning of this2′-modification in the Dicer-resistant hairpin structure, thus enablingone to design better RNAi constructs with less or no off-targetsilencing.

In one embodiment, a hairpin polynucleotide of the invention cancomprise one nucleic acid portion which is DNA and one nucleic acidportion which is RNA. Antisense (guide) sequences of the invention canbe “chimeric oligonucleotides” which comprise an RNA-like and a DNA-likeregion.

The language “RNase H activating region” includes a region of anoligonucleotide, e.g., a chimeric oligonucleotide, that is capable ofrecruiting RNase H to cleave the target RNA strand to which theoligonucleotide binds. Typically, the RNase activating region contains aminimal core (of at least about 3-5, typically between about 3-12, moretypically, between about 5-12, and more preferably between about 5-10contiguous nucleomonomers) of DNA or DNA-like nucleomonomers. (See,e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activatingregion comprises about nine contiguous deoxyribose containingnucleomonomers.

The language “non-activating region” includes a region of an antisensesequence, e.g., a chimeric oligonucleotide, that does not recruit oractivate RNase H. Preferably, a non-activating region does not comprisephosphorothioate DNA. The oligonucleotides of the invention comprise atleast one non-activating region. In one embodiment, the non-activatingregion can be stabilized against nucleases or can provide specificityfor the target by being complementary to the target and forming hydrogenbonds with the target nucleic acid molecule, which is to be bound by theoligonucleotide.

In one embodiment, at least a portion of the contiguous polynucleotidesare linked by a substitute linkage, e.g., a phosphorothioate linkage.

In certain embodiments, most or all of the nucleotides beyond the guidesequence (2′-modified or not) are linked by phosphorothioate linkages.Such constructs tend to have improved pharmacokinetics due to theirhigher affinity for serum proteins. The phosphorothioate linkages in thenon-guide sequence portion of the polynucleotide generally do notinterfere with guide strand activity, once the latter is loaded intoRISC.

Antisense (guide) sequences of the present invention may include“morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionicand function by an RNase H-independent mechanism. Each of the 4 geneticbases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholinooligonucleotides is linked to a 6-membered morpholine ring. Morpholinooligonucleotides are made by joining the 4 different subunit types by,e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholinooligonucleotides have many advantages including: complete resistance tonucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267); predictabletargeting (Biochemica Biophysica Acta. 1999. 1489:141); reliableactivity in cells (Antisense & Nucl. Acid Drug Dev. 1997. 7:63);excellent sequence to specificity (Antisense & Nucl. Acid Drug Dev.1997. 7:151); minimal non-antisense activity (Biochemica BiophysicaActa. 1999. 1489:141); and simple osmotic or scrape delivery (Antisense& Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides arealso preferred because of their non-toxicity at high doses. A discussionof the preparation of morpholino oligonucleotides can be found inAntisense & Nucl. Acid Drug Dev. 1997. 7:187.

The chemical modifications described herein are believed, based on thedata described herein, to promote single stranded polynucleotide loadinginto the RISC. Single stranded polynucleotides have been shown to beactive in loading into RISC and inducing gene silencing. However, thelevel of activity for single stranded polynucleotides appears to be 2 to4 orders of magnitude lower when compared to a duplex polynucleotide.

The present invention provides a description of the chemicalmodification patterns, which may (a) significantly increase stability ofthe single stranded polynucleotide (b) promote efficient loading of thepolynucleotide into the RISC complex and (c) improve uptake of thesingle stranded nucleotide by the cell. FIG. 18 provides somenon-limiting examples of the chemical modification patterns which may bebeneficial for achieving single stranded polynucleotide efficacy insidethe cell. The chemical modification patterns may include combination ofribose, backbone, hydrophobic nucleoside and conjugate type ofmodifications. In addition, in some of the embodiments, the 5′ end ofthe single polynucleotide may be chemically phosphorylated.

In yet another embodiment, the present invention provides a descriptionof the chemical modifications patterns, which improve functionality ofRISC inhibiting polynucleotides. Single stranded polynucleotides havebeen shown to inhibit activity of a preloaded RISC complex through thesubstrate competition mechanism. For these types of molecules,conventionally called antagomers, the activity usually requires highconcentration and in vivo delivery is not very effective. The presentinvention provides a description of the chemical modification patterns,which may (a) significantly increase stability of the single strandedpolynucleotide (b) promote efficient recognition of the polynucleotideby the RISC as a substrate and/or (c) improve uptake of the singlestranded nucleotide by the cell. FIG. 6 provides some non-limitingexamples of the chemical modification patterns that may be beneficialfor achieving single stranded polynucleotide efficacy inside the cell.The chemical modification patterns may include combination of ribose,backbone, hydrophobic nucleoside and conjugate type of modifications.

The modifications provided by the present invention are applicable toall polynucleotides. This includes single stranded RISC enteringpolynucleotides, single stranded RISC inhibiting polynucleotides,conventional duplexed polynucleotides of variable length (15-40 bp),asymmetric duplexed polynucleotides, and the like. Polynucleotides maybe modified with wide variety of chemical modification patterns,including 5′ end, ribose, backbone and hydrophobic nucleosidemodifications.

Synthesis

Oligonucleotides of the invention can be synthesized by any method knownin the art, e.g., using enzymatic synthesis and/or chemical synthesis.The oligonucleotides can be synthesized in vitro (e.g., using enzymaticsynthesis and chemical synthesis) or in vivo (using recombinant DNAtechnology well known in the art).

In a preferred embodiment, chemical synthesis is used for modifiedpolynucleotides. Chemical synthesis of linear oligonucleotides is wellknown in the art and can be achieved by solution or solid phasetechniques. Preferably, synthesis is by solid phase methods.Oligonucleotides can be made by any of several different syntheticprocedures including the phosphoramidite, phosphite triester,H-phosphonate, and phosphotriester methods, typically by automatedsynthesis methods.

Oligonucleotide synthesis protocols are well known in the art and can befound, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al. 1984.J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908;Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl.Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook ofBiochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.;Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S.Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J.Med. Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; and U.S. Pat.No. 5,264,423.

The synthesis method selected can depend on the length of the desiredoligonucleotide and such choice is within the skill of the ordinaryartisan. For example, the phosphoramidite and phosphite triester methodcan produce oligonucleotides having 175 or more nucleotides, while theH-phosphonate method works well for oligonucleotides of less than 100nucleotides. If modified bases are incorporated into theoligonucleotide, and particularly if modified phosphodiester linkagesare used, then the synthetic procedures are altered as needed accordingto known procedures. In this regard, Uhlmann et al. (1990, ChemicalReviews 90:543-584) provide references and outline procedures for makingoligonucleotides with modified bases and modified phosphodiesterlinkages. Other exemplary methods for making oligonucleotides are taughtin Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”;Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methodsare also taught in “Oligonucleotide Synthesis—A Practical Approach”(Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover,linear oligonucleotides of defined sequence, including some sequenceswith modified nucleotides, are readily available from several commercialsources.

The oligonucleotides may be purified by polyacrylamide gelelectrophoresis, or by any of a number of chromatographic methods,including gel chromatography and high pressure liquid chromatography. Toconfirm a nucleotide sequence, especially unmodified nucleotidesequences, oligonucleotides may be subjected to DNA sequencing by any ofthe known procedures, including Maxam and Gilbert sequencing, Sangersequencing, capillary electrophoresis sequencing, the wandering spotsequencing procedure or by using selective chemical degradation ofoligonucleotides bound to Hybond paper. Sequences of shortoligonucleotides can also be analyzed by laser desorption massspectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am.Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom.14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencingmethods are also available for RNA oligonucleotides.

The quality of oligonucleotides synthesized can be verified by testingthe oligonucleotide by capillary electrophoresis and denaturing stronganion HPLC (SAX-HPLC) using, e.g., the method of Bergot and Egan. 1992.J. Chrom. 599:35.

Other exemplary synthesis techniques are well known in the art (see,e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, SecondEdition (1989); DNA Cloning, Volumes I and II (DN Glover Ed. 1985);Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation(B D Hames and S J Higgins eds. 1984); A Practical Guide to MolecularCloning (1984); or the series, Methods in Enzymology (Academic Press,Inc.)).

In certain embodiments, the subject RNAi constructs or at least portionsthereof are transcribed from expression vectors encoding the subjectconstructs. Any art recognized vectors may be use for this purpose. Thetranscribed RNAi constructs may be isolated and purified, before desiredmodifications (such as replacing an unmodified sense strand with amodified one, etc.) are carried out.

Delivery/Carrier Uptake of Oligonucleotides by Cells

Oligonucleotides and oligonucleotide compositions are contacted with(i.e., brought into contact with, also referred to herein asadministered or delivered to) and taken up by one or more cells or acell lysate. The term “cells” includes prokaryotic and eukaryotic cells,preferably vertebrate cells, and, more preferably, mammalian cells. In apreferred embodiment, the oligonucleotide compositions of the inventionare contacted with human cells.

Oligonucleotide compositions of the invention can be contacted withcells in vitro, e.g., in a test tube or culture dish, (and may or maynot be introduced into a subject) or in vivo, e.g., in a subject such asa mammalian subject. In some embodiments, Oligonucleotides areadministered topically or through electroporation. Oligonucleotides aretaken up by cells at a slow rate by endocytosis, but endocytosedoligonucleotides are generally sequestered and not available, e.g., forhybridization to a target nucleic acid molecule. In one embodiment,cellular uptake can be facilitated by electroporation or calciumphosphate precipitation. However, these procedures are only useful forin vitro or ex vivo embodiments, are not convenient and, in some cases,are associated with cell toxicity.

In another embodiment, delivery of oligonucleotides into cells can beenhanced by suitable art recognized methods including calcium phosphate,DMSO, glycerol or dextran, electroporation, or by transfection, e.g.,using cationic, anionic, or neutral lipid compositions or liposomesusing methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic AcidsResearch. 21:3567). Enhanced delivery of oligonucleotides can also bemediated by the use of vectors (See e.g., Shi, Y. 2003. Trends Genet.2003 Jan. 19:9; Reichhart J. Metal. Genesis. 2002. 34(1-2):1604, Yu etal. 2002. Proc. Natl. Acad. Sci. USA 99:6047; Sui et al. 2002. Proc.Natl. Acad. Sci. USA 99:5515) viruses, polyamine or polycationconjugates using compounds such as polylysine, protamine, or Ni,N12-bis(ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989.Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl.Acad. Sci. 88:4255).

In certain embodiments, the sd-rxRNA of the invention may be deliveredby using various beta-glucan containing particles, referred to as GeRPs(glucan encapsulated RNA loaded particle), described in, andincorporated by reference from, U.S. Provisional Application No.61/310,611, filed on Mar. 4, 2010 and entitled “Formulations and Methodsfor Targeted Delivery to Phagocyte Cells.” Such particles are alsodescribed in, and incorporated by reference from US Patent PublicationsUS 2005/0281781 A1, and US 2010/0040656, and in PCT publications WO2006/007372, and WO 2007/050643. The sd-rxRNA molecule may behydrophobically modified and optionally may be associated with a lipidand/or amphiphilic peptide. In certain embodiments, the beta-glucanparticle is derived from yeast. In certain embodiments, the payloadtrapping molecule is a polymer, such as those with a molecular weight ofat least about 1000 Da, 10,000 Da, 50,000 Da, 100 kDa, 500 kDa, etc.Preferred polymers include (without limitation) cationic polymers,chitosans, or PEI (polyethylenimine), etc.

Glucan particles can be derived from insoluble components of fungal cellwalls such as yeast cell walls. In some embodiments, the yeast isBaker's yeast. Yeast-derived glucan molecules can include one or more ofB-(1,3)-Glucan, B-(1,6)-Glucan, mannan and chitin. In some embodiments,a glucan particle comprises a hollow yeast cell wall whereby theparticle maintains a three dimensional structure resembling a cell,within which it can complex with or encapsulate a molecule such as anRNA molecule. Some of the advantages associated with the use of yeastcell wall particles are availability of the components, theirbiodegradable nature, and their ability to be targeted to phagocyticcells.

In some embodiments, glucan particles can be prepared by extraction ofinsoluble components from cell walls, for example by extracting Baker'syeast (Fleischmann's) with 1M NaOH/pH 4.0 H2O, followed by washing anddrying. Methods of preparing yeast cell wall particles are discussed in,and incorporated by reference from U.S. Pat. Nos. 4,810,646, 4,992,540,5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079,5,607,677, 5,968,811, 6,242,594, 6,444,448, 6,476,003, US PatentPublications 2003/0216346, 2004/0014715 and 2010/0040656, and PCTpublished application WO02/12348.

Protocols for preparing glucan particles are also described in, andincorporated by reference from, the following references: Soto andOstroff (2008), “Characterization of multilayered nanoparticlesencapsulated in yeast cell wall particles for DNA delivery.” BioconjugChem 19(4):840-8; Soto and Ostroff (2007), “Oral Macrophage MediatedGene Delivery System,” Nanotech, Volume 2, Chapter 5 (“Drug Delivery”),pages 378-381; and Li et al. (2007), “Yeast glucan particles activatemurine resident macrophages to secrete proinflammatory cytokines viaMyD88- and Syk kinase-dependent pathways.” Clinical Immunology124(2):170-181.

Glucan containing particles such as yeast cell wall particles can alsobe obtained to commercially. Several non-limiting examples include:Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAFAgri, Minneapolis, Minn.), Nutrex (Sensient Technologies, Milwaukee,Wis.), alkali-extracted particles such as those produced by Nutricepts(Nutricepts Inc., Burnsville, Minn.) and ASA Biotech, acid-extracted WGPparticles from Biopolymer Engineering, and organic solvent-extractedparticles such as Adjuvax™ from Alpha-beta Technology, Inc. (Worcester,Mass.) and microparticulate glucan from Novogen (Stamford, Conn.).

Glucan particles such as yeast cell wall particles can have varyinglevels of purity depending on the method of production and/orextraction. In some instances, particles are alkali-extracted,acid-extracted or organic solvent-extracted to remove intracellularcomponents and/or the outer mannoprotein layer of the cell wall. Suchprotocols can produce particles that have a glucan (w/w) content in therange of 50%-90%. In some instances, a particle of lower purity, meaninglower glucan w/w content may be preferred, while in other embodiments, aparticle of higher purity, meaning higher glucan w/w content may bepreferred.

Glucan particles, such as yeast cell wall particles, can have a naturallipid content. For example, the particles can contain 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20% or more than 20% w/w lipid. In the Examples section, theeffectiveness of two glucan particle batches are tested: YGP SAF and YGPSAF+L (containing natural lipids). In some instances, the presence ofnatural lipids may assist in complexation or capture of RNA molecules.

Glucan containing particles typically have a diameter of approximately2-4 microns, although particles with a diameter of less than 2 micronsor greater than 4 microns are also compatible with aspects of theinvention.

The RNA molecule(s) to be delivered are complexed or “trapped” withinthe shell of the glucan particle. The shell or RNA component of theparticle can be labeled for visualization, as described in, andincorporated by reference from, Soto and Ostroff (2008) Bioconjug Chem19:840. Methods of loading GeRPs are discussed further below.

The optimal protocol for uptake of oligonucleotides will depend upon anumber of factors, the most crucial being the type of cells that arebeing used. Other factors that are important in uptake include, but arenot limited to, the nature and concentration of the oligonucleotide, theconfluence of the cells, the type of culture the cells are in (e.g., asuspension culture or plated) and the type of media in which the cellsare grown.

Encapsulating Agents

Encapsulating agents entrap oligonucleotides within vesicles. In anotherembodiment of the invention, an oligonucleotide may be associated with acarrier or vehicle, e.g., liposomes or micelles, although other carrierscould be used, as would be appreciated by one skilled in the art.Liposomes are vesicles made of a lipid bilayer having a structuresimilar to biological membranes. Such carriers are used to facilitatethe cellular uptake or targeting of the oligonucleotide, or improve theoligonucleotide's pharmacokinetic or toxicologic properties.

For example, the oligonucleotides of the present invention may also beadministered encapsulated in liposomes, pharmaceutical compositionswherein the active ingredient is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentric layers adherentto lipidic layers. The oligonucleotides, depending upon solubility, maybe present both in the aqueous layer and in the lipidic layer, or inwhat is generally termed a liposomic suspension. The hydrophobic layer,generally but not exclusively, comprises phopholipids such as lecithinand sphingomyelin, steroids such as cholesterol, more or less ionicsurfactants such as diacetylphosphate, stearylamine, or phosphatidicacid, or other materials of a hydrophobic nature. The diameters of theliposomes generally range from about 15 nm to about 5 microns.

The use of liposomes as drug delivery vehicles offers severaladvantages. Liposomes increase intracellular stability, increase uptakeefficiency and improve biological activity. Liposomes are hollowspherical vesicles composed of lipids arranged in a similar fashion asthose lipids which make up the cell membrane. They have an internalaqueous space for entrapping water soluble compounds and range in sizefrom 0.05 to several microns in diameter. Several studies have shownthat liposomes can deliver nucleic acids to cells and that the nucleicacids remain biologically active. For example, a lipid delivery vehicleoriginally designed as a research tool, such as Lipofectin orLIPOFECTAMINE™ 2000, can deliver intact nucleic acid molecules to cells.

Specific advantages of using liposomes include the following: they arenon-toxic and biodegradable in composition; they display longcirculation half-lives; and recognition molecules can be readilyattached to their surface for targeting to tissues. Finally,cost-effective manufacture of liposome-based pharmaceuticals, either ina liquid suspension or lyophilized product, has demonstrated theviability of this technology as an acceptable drug delivery system.

In some aspects, formulations associated with the invention might beselected for a class of naturally occurring or chemically synthesized ormodified saturated and unsaturated fatty acid residues. Fatty acidsmight exist in a form of triglycerides, diglycerides or individual fattyacids. In another embodiment, the use of well-validated mixtures offatty acids and/or fat emulsions currently used in pharmacology forparenteral nutrition may be utilized.

Liposome based formulations are widely used for oligonucleotidedelivery. However, most of commercially available lipid or liposomeformulations contain at least one positively charged lipid (cationiclipids). The presence of this positively charged lipid is believed to beessential for obtaining a high degree of oligonucleotide loading and forenhancing liposome fusogenic properties. Several methods have beenperformed and published to identify optimal positively charged lipidchemistries. However, the commercially available liposome formulationscontaining cationic lipids are characterized by a high level oftoxicity. In vivo limited therapeutic indexes have revealed thatliposome formulations containing positive charged lipids are associatedwith toxicity (i.e. elevation in liver enzymes) at concentrations onlyslightly higher than concentration required to achieve RNA silencing.

Nucleic acids associated with the invention can be hydrophobicallymodified and can be encompassed within neutral nanotransporters. Furtherdescription of neutral nanotransporters is incorporated by referencefrom PCT Application PCT/US2009/005251, filed on Sep. 22, 2009, andentitled “Neutral Nanotransporters.” Such particles enable quantitativeoligonucleotide incorporation into non-charged lipid mixtures. The lackof toxic levels of cationic lipids in such neutral nanotransportercompositions is an important feature.

As demonstrated in PCT/US2009/005251, oligonucleotides can effectivelybe incorporated into a lipid mixture that is free of cationic lipids andsuch a composition can effectively deliver a therapeutic oligonucleotideto a cell in a manner that it is functional. For example, a high levelof activity was observed when the fatty mixture was composed of aphosphatidylcholine base fatty acid and a sterol such as a cholesterol.For instance, one preferred formulation of neutral fatty mixture iscomposed of at least 20% of DOPC or DSPC and at least 20% of sterol suchas cholesterol. Even as low as 1:5 lipid to oligonucleotide ratio wasshown to be sufficient to get complete encapsulation of theoligonucleotide in a non charged formulation.

The neutral nanotransporters compositions enable efficient loading ofoligonucleotide into neutral fat formulation. The composition includesan oligonucleotide that is modified in a manner such that thehydrophobicity of the molecule is increased (for example a hydrophobicmolecule is attached (covalently or no-covalently) to a hydrophobicmolecule on the oligonucleotide terminus or a non-terminal nucleotide,base, sugar, or backbone), the modified oligonucleotide being mixed witha neutral fat formulation (for example containing at least 25% ofcholesterol and 25% of DOPC or analogs thereof). A cargo molecule, suchas another lipid can also be included in the composition. Thiscomposition, where part of the formulation is build into theoligonucleotide itself, enables efficient encapsulation ofoligonucleotide in neutral lipid particles.

In some aspects, stable particles ranging in size from 50 to 140 nm canbe formed upon complexing of hydrophobic oligonucleotides with preferredformulations. It is interesting to mention that the formulation byitself typically does not form small particles, but rather, formsagglomerates, which are transformed into stable 50-120 nm particles uponaddition of the hydrophobic modified oligonucleotide.

The neutral nanotransporter compositions of the invention include ahydrophobic modified polynucleotide, a neutral fatty mixture, andoptionally a cargo molecule. A “hydrophobic modified polynucleotide” asused herein is a polynucleotide of the invention (i.e. sd-rxRNA) thathas at least one modification that renders the polynucleotide morehydrophobic than the polynucleotide was prior to modification. Themodification may be achieved by attaching (covalently or non-covalently)a hydrophobic molecule to the polynucleotide. In some instances thehydrophobic molecule is or includes a lipophilic group.

The term “lipophilic group” means a group that has a higher affinity forlipids than its affinity for water. Examples of lipophilic groupsinclude, but are not limited to, cholesterol, a cholesteryl or modifiedcholesteryl residue, adamantine, dihydrotesterone, long chain alkyl,long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic,oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholicacid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoylcholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids,such as steroids, vitamins, such as vitamin E, fatty acids eithersaturated or unsaturated, fatty acid esters, such as triglycerides,pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin,coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. Thecholesterol moiety may be reduced (e.g. as in cholestan) or may besubstituted (e.g. by halogen). A combination of different lipophilicgroups in one molecule is also possible.

The hydrophobic molecule may be attached at various positions of thepolynucleotide. As described above, the hydrophobic molecule may belinked to the terminal residue of the polynucleotide such as the 3′ of5′-end of the polynucleotide. Alternatively, it may be linked to aninternal nucleotide or a nucleotide on a branch of the polynucleotide.The hydrophobic molecule may be attached, for instance to a 2′-positionof the nucleotide. The hydrophobic molecule may also be linked to theheterocyclic base, the sugar or the backbone of a nucleotide of thepolynucleotide.

The hydrophobic molecule may be connected to the polynucleotide by alinker moiety. Optionally the linker moiety is a non-nucleotidic linkermoiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer),oligoethyleneglycol, such as triethyleneglycol (spacer 9) orhexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol. Thespacer units are preferably linked by phosphodiester or phosphorothioatebonds. The linker units may appear just once in the molecule or may beincorporated several times, e.g. via phosphodiester, phosphorothioate,methylphosphonate, or amide linkages.

Typical conjugation protocols involve the synthesis of polynucleotidesbearing an aminolinker at one or more positions of the sequence,however, a linker is not required. The amino group is then reacted withthe molecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with thepolynucleotide still bound to a solid support or following cleavage ofthe polynucleotide in solution phase. Purification of the modifiedpolynucleotide by HPLC typically results in a pure material.

In some embodiments the hydrophobic molecule is a sterol type conjugate,a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugatewith altered side chain length, fatty acid conjugate, any otherhydrophobic group conjugate, and/or hydrophobic modifications of theinternal nucleoside, which provide sufficient hydrophobicity to beincorporated into micelles.

For purposes of the present invention, the term “sterols”, refers orsteroid alcohols are a subgroup of steroids with a hydroxyl group at the3-position of the A-ring. They are amphipathic lipids synthesized fromacetyl-coenzyme A via the HMG-CoA reductase pathway. The overallmolecule is quite flat. The hydroxyl group on the A ring is polar. Therest of the aliphatic chain is non-polar. Usually sterols are consideredto have an 8 carbon chain at position 17.

For purposes of the present invention, the term “sterol type molecules”,refers to steroid alcohols, which are similar in structure to sterols.The main difference is the structure of the ring and number of carbonsin a position 21 attached side chain.

For purposes of the present invention, the term “PhytoSterols” (alsocalled plant sterols) are a group of steroid alcohols, phytochemicalsnaturally occurring in plants. There are more then 200 different knownPhytoSterols

For purposes of the present invention, the term “Sterol side chain”refers to a chemical composition of a side chain attached at theposition 17 of sterol-type molecule. In a standard definition sterolsare limited to a 4 ring structure carrying a 8 carbon chain at position17. In this invention, the sterol type molecules with side chain longerand shorter than conventional are described. The side chain may branchedor contain double back bones.

Thus, sterols useful in the invention, for example, includecholesterols, as well as unique sterols in which position 17 hasattached side chain of 2-7 or longer then 9 carbons. In a particularembodiment, the length of the polycarbon tail is varied between 5 and 9carbons. Such conjugates may have significantly better in vivo efficacy,in particular delivery to liver. These types of molecules are expectedto work at concentrations 5 to 9 fold lower then oligonucleotidesconjugated to conventional cholesterols.

Alternatively the polynucleotide may be bound to a protein, peptide orpositively charged chemical that functions as the hydrophobic molecule.The proteins may be selected from the group consisting of protamine,dsRNA binding domain, and arginine rich peptides. Exemplary positivelycharged chemicals include spermine, spermidine, cadaverine, andputrescine.

In another embodiment hydrophobic molecule conjugates may demonstrateeven higher efficacy when it is combined with optimal chemicalmodification patterns of the polynucleotide (as described herein indetail), containing but not limited to hydrophobic modifications,phosphorothioate modifications, and 2′ ribo modifications.

In another embodiment the sterol type molecule may be a naturallyoccurring PhytoSterols. The polycarbon chain may be longer than 9 andmay be linear, branched and/or contain double bonds. Some PhytoSterolcontaining polynucleotide conjugates may be significantly more potentand active in delivery of polynucleotides to various tissues. SomePhytoSterols may demonstrate tissue preference and thus be used as a wayto delivery RNAi specifically to particular tissues.

The hydrophobic modified polynucleotide is mixed with a neutral fattymixture to form a micelle. The neutral fatty acid mixture is a mixtureof fats that has a net neutral or slightly net negative charge at oraround physiological pH that can form a micelle with the hydrophobicmodified polynucleotide. For purposes of the present invention, the term“micelle” refers to a small nanoparticle formed by a mixture of noncharged fatty acids and phospholipids. The neutral fatty mixture mayinclude cationic lipids as long as they are present in an amount thatdoes not cause toxicity. In preferred embodiments the neutral fattymixture is free of cationic lipids. A mixture that is free of cationiclipids is one that has less than 1% and preferably 0% of the total lipidbeing cationic lipid. The term “cationic lipid” includes lipids andsynthetic lipids having a net positive charge at or around physiologicalpH. The term “anionic lipid” includes lipids and synthetic lipids havinga net negative charge at or around physiological pH.

The neutral fats bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction).

The neutral fat mixture may include formulations selected from a classof naturally occurring or chemically synthesized or modified saturatedand unsaturated fatty acid residues. Fatty acids might exist in a formof triglycerides, diglycerides or individual fatty acids. In anotherembodiment the use of well-validated mixtures of fatty acids and/or fatemulsions currently used in pharmacology for parenteral nutrition may beutilized.

The neutral fatty mixture is preferably a mixture of a choline basedfatty acid and a sterol. Choline based fatty acids include for instance,synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC,DSPC, DOPC, POPC, and DEPC. DOPC (chemical registry number 4235-95-4) isdioleoylphosphatidylcholine (also known asdielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine,dioleoyl-sn-glycero-3-phosphocholine, dioleylphosphatidylcholine). DSPC(chemical registry number 816-94-4) is distearoylphosphatidylcholine(also known as 1,2-Distearoyl-sn-Glycero-3-phosphocholine).

The sterol in the neutral fatty mixture may be for instance cholesterol.The neutral fatty mixture may be made up completely of a choline basedfatty acid and a sterol or it may optionally include a cargo molecule.For instance, the neutral fatty mixture may have at least 20% or 25%fatty acid and 20% or 25% sterol.

For purposes of the present invention, the term “Fatty acids” relates toconventional description of fatty acid. They may exist as individualentities or in a form of two- and triglycerides. For purposes of thepresent invention, the term “fat emulsions” refers to safe fatformulations given intravenously to subjects who are unable to getenough fat in their diet. It is an emulsion of soy bean oil (or othernaturally occurring oils) and egg phospholipids. Fat emulsions are beingused for formulation of some insoluble anesthetics. In this disclosure,fat emulsions might be part of commercially available preparations likeIntralipid, Liposyn, Nutrilipid, modified commercial preparations, wherethey are enriched with particular fatty acids or fully denovo-formulated combinations of fatty acids and phospholipids.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days.

50%-60% of the formulation can optionally be any other lipid ormolecule. Such a lipid or molecule is referred to herein as a cargolipid or cargo molecule. Cargo molecules include but are not limited tointralipid, small molecules, fusogenic peptides or lipids or other smallmolecules might be added to alter cellular uptake, endosomal release ortissue distribution properties. The ability to tolerate cargo moleculesis important for modulation of properties of these particles, if suchproperties are desirable. For instance the presence of some tissuespecific metabolites might drastically alter tissue distributionprofiles. For example use of Intralipid type formulation enriched inshorter or longer fatty chains with various degrees of saturationaffects tissue distribution profiles of these type of formulations (andtheir loads).

An example of a cargo lipid useful according to the invention is afusogenic lipid. For to instance, the zwiterionic lipid DOPE (chemicalregistry number 4004-5-1,1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine)is a preferred cargo lipid.

Intralipid may be comprised of the following composition: 1 000 mLcontain: purified soybean oil 90 g, purified egg phospholipids 12 g,glycerol anhydrous 22 g, water for injection q.s. ad 1 000 mL. pH isadjusted with sodium hydroxide to pH approximately 8. Energy content/L:4.6 MJ (190 kcal). Osmolality (approx.): 300 mOsm/kg water. In anotherembodiment fat emulsion is Liposyn that contains 5% safflower oil, 5%soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5%glycerin in water for injection. It may also contain sodium hydroxidefor pH adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolarity of 276 mOsmol/liter (actual).

Variation in the identity, amounts and ratios of cargo lipids affectsthe cellular uptake and tissue distribution characteristics of thesecompounds. For example, the length of lipid tails and level ofsaturability will affect differential uptake to liver, lung, fat andcardiomyocytes. Addition of special hydrophobic molecules like vitaminsor different forms of sterols can favor distribution to special tissueswhich are involved in the metabolism of particular compounds. In someembodiments, vitamin A or E is used. Complexes are formed at differentoligonucleotide concentrations, with higher concentrations favoring moreefficient complex formation.

In another embodiment, the fat emulsion is based on a mixture of lipids.Such lipids may include natural compounds, chemically synthesizedcompounds, purified fatty acids or any other lipids. In yet anotherembodiment the composition of fat emulsion is entirely artificial. In aparticular embodiment, the fat emulsion is more then 70% linoleic acid.In yet another particular embodiment the fat emulsion is at least 1% ofcardiolipin. Linoleic acid (LA) is an unsaturated omega-6 fatty acid. Itis a colorless liquid made of a carboxylic acid with an 18-carbon chainand two cis double bonds.

In yet another embodiment of the present invention, the alteration ofthe composition of the fat emulsion is used as a way to alter tissuedistribution of hydrophobicly modified polynucleotides. This methodologyprovides for the specific delivery of the polynucleotides to particulartissues.

In another embodiment the fat emulsions of the cargo molecule containmore then 70% of Linoleic acid (C18H32O2) and/or cardiolipin.

Fat emulsions, like intralipid have been used before as a deliveryformulation for some non-water soluble drugs (such as Propofol,re-formulated as Diprivan). Unique features of the present inventioninclude (a) the concept of combining modified polynucleotides with thehydrophobic compound(s), so it can be incorporated in the fat micellesand (b) mixing it with the fat emulsions to provide a reversiblecarrier. After injection into a blood stream, micelles usually bind toserum proteins, including albumin, HDL, LDL and other. This binding isreversible and eventually the fat is absorbed by cells. Thepolynucleotide, incorporated as a part of the micelle will then bedelivered closely to the surface of the cells. After that cellularuptake might be happening though variable mechanisms, including but notlimited to sterol type delivery.

Complexing Agents

Complexing agents bind to the oligonucleotides of the invention by astrong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction). In one embodiment,oligonucleotides of the invention can be complexed with a complexingagent to increase cellular uptake of oligonucleotides. An example of acomplexing agent includes cationic lipids. Cationic lipids can be usedto deliver oligonucleotides to cells. However, as discussed above,formulations free in cationic lipids are preferred in some embodiments.

The term “cationic lipid” includes lipids and synthetic lipids havingboth polar and non-polar domains and which are capable of beingpositively charged at or around physiological pH and which bind topolyanions, such as nucleic acids, and facilitate the delivery ofnucleic acids into cells. In general cationic lipids include saturatedand unsaturated alkyl and alicyclic ethers and esters of amines, amides,or derivatives thereof. Straight-chain and branched alkyl and alkenylgroups of cationic lipids can contain, e.g., from 1 to about 25 carbonatoms. Preferred straight chain or branched alkyl or alkene groups havesix or more carbon atoms. Alicyclic groups include cholesterol and othersteroid groups. Cationic lipids can be prepared with a variety ofcounterions (anions) including, e.g., Cl⁻, Br⁻, I⁻, F⁻, acetate,trifluoroacetate, sulfate, nitrite, and nitrate.

Examples of cationic lipids include polyethylenimine, polyamidoamine(PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA andDOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE,Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL,San Luis Obispo, Calif.). Exemplary cationic liposomes can be made fromN-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3β-N—(N′,N′-dimethylaminoethane)carbamoyllcholesterol(DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). The cationic lipidN-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),for example, was found to increase 1000-fold the antisense effect of aphosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Oligonucleotides can also be complexedwith, e.g., poly (L-lysine) or avidin and lipids may, or may not, beincluded in this mixture, e.g., steryl-poly (L-lysine).

Cationic lipids have been used in the art to deliver oligonucleotides tocells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430;5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipidcompositions which can be used to facilitate uptake of the instantoligonucleotides can be used in connection with the claimed methods. Inaddition to those listed supra, other lipid compositions are also knownin the art and include, e.g., those taught in U.S. Pat. No. 4,235,871;U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

In one embodiment lipid compositions can further comprise agents, e.g.,viral proteins to enhance lipid-mediated transfections ofoligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). Inanother embodiment, oligonucleotides are contacted with cells as part ofa composition comprising an oligonucleotide, a peptide, and a lipid astaught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also beendescribed which are serum resistant (Lewis, et al., 1996. Proc. Natl.Acad. Sci. 93:3176). Cationic lipids and other complexing agents act toincrease the number of oligonucleotides carried into the cell throughendocytosis.

In another embodiment N-substituted glycine oligonucleotides (peptoids)can be used to optimize uptake of oligonucleotides. Peptoids have beenused to create cationic lipid-like compounds for transfection (Murphy,et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can besynthesized using standard methods (e.g., Zuckermann, R. N., et al.1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int.J. Peptide Protein Res. 40:497). Combinations of cationic lipids andpeptoids, liptoids, can also be used to optimize uptake of the subjectoligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345).Liptoids can be synthesized by elaborating peptoid oligonucleotides andcoupling the amino terminal submonomer to a lipid via its amino group(Hunag, et al., 1998. Chemistry and Biology. 5:345).

It is known in the art that positively charged amino acids can be usedfor creating highly active cationic lipids (Lewis et al. 1996. Proc.Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a composition fordelivering oligonucleotides of the invention comprises a number ofarginine, lysine, histidine or ornithine residues linked to a lipophilicmoiety (see e.g., U.S. Pat. No. 5,777,153).

In another embodiment, a composition for delivering oligonucleotides ofthe invention comprises a peptide having from between about one to aboutfour basic residues. These basic residues can be located, e.g., on theamino terminal, C-terminal, or internal region of the peptide. Familiesof amino acid residues having similar side chains have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine (canalso be considered non-polar), asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Apart from the basic amino acids, a majority or all of theother residues of the peptide can be selected from the non-basic aminoacids, e.g., amino acids other than lysine, arginine, or histidine.Preferably a preponderance of neutral amino acids with long neutral sidechains are used.

In one embodiment, a composition for delivering oligonucleotides of theinvention comprises a natural or synthetic polypeptide having one ormore gamma carboxyglutamic acid residues, or γ-Gla residues. These gammacarboxyglutamic acid residues may enable the polypeptide to bind to eachother and to membrane surfaces. In other words, a polypeptide having aseries of γ-Gla may be used as a general delivery modality that helps anRNAi construct to stick to whatever membrane to which it comes incontact. This may at least slow RNAi constructs from being cleared fromthe blood stream and enhance their chance of homing to the target.

The gamma carboxyglutamic acid residues may exist in natural proteins(for example, prothrombin has 10 γ-Gla residues). Alternatively, theycan be introduced into the purified, recombinantly produced, orchemically synthesized polypeptides by carboxylation using, for example,a vitamin K-dependent carboxylase. The gamma carboxyglutamic acidresidues may be consecutive or non-consecutive, and the total number andlocation of such gamma carboxyglutamic acid residues in the polypeptidecan be regulated/fine tuned to achieve different levels of “stickiness”of the polypeptide.

In one embodiment, the cells to be contacted with an oligonucleotidecomposition of the invention are contacted with a mixture comprising theoligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 12 hoursto about 24 hours. In another embodiment, the cells to be contacted withan oligonucleotide composition are contacted with a mixture comprisingthe oligonucleotide and a mixture comprising a lipid, e.g., one of thelipids or lipid compositions described supra for between about 1 andabout five days. In one embodiment, the cells are contacted with amixture comprising a lipid and the oligonucleotide for between aboutthree days to as long as about 30 days. In another embodiment, a mixturecomprising a lipid is left in contact with the cells for at least aboutfive to about 20 days. In another embodiment, a mixture comprising alipid is left in contact with the cells for at least about seven toabout 15 days.

For example, in one embodiment, an oligonucleotide composition can becontacted with cells in the presence of a lipid such as cytofectin CS orGSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 forprolonged incubation periods as described herein.

In one embodiment, the incubation of the cells with the mixturecomprising a lipid and an oligonucleotide composition does not reducethe viability of the cells. Preferably, after the transfection periodthe cells are substantially viable. In one embodiment, aftertransfection, the cells are between at least about 70% and at leastabout 100% viable. In another embodiment, the cells are between at leastabout 80% and at least about 95% viable. In yet another embodiment, thecells are between at least about 85% and at least about 90% viable.

In one embodiment, oligonucleotides are modified by attaching a peptidesequence that transports the oligonucleotide into a cell, referred toherein as a “transporting peptide.” In one embodiment, the compositionincludes an oligonucleotide which is complementary to a target nucleicacid molecule encoding the protein, and a covalently attachedtransporting peptide.

The language “transporting peptide” includes an amino acid sequence thatfacilitates the transport of an oligonucleotide into a cell. Exemplarypeptides which facilitate the transport of the moieties to which theyare linked into cells are known in the art, and include, e.g., HIV TATtranscription factor, lactoferrin, Herpes VP22 protein, and fibroblastgrowth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; andDerossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare.1997. Cell 88:223).

Oligonucleotides can be attached to the transporting peptide using knowntechniques, e.g., (Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629;Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J.Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem. 272:16010). Forexample, in one embodiment, oligonucleotides bearing an activated thiolgroup are linked via that thiol group to a cysteine present in atransport peptide (e.g., to the cysteine present in the (3 turn betweenthe second and the third helix of the antennapedia homeodomain astaught, e.g., in Derossi et al. 1998. Trends Cell Biol. 8:84;Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al.1995. J. Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OHgroup can be coupled to the transport peptide as the last (N-terminal)amino acid and an oligonucleotide bearing an SH group can be coupled tothe peptide (Troy et al. 1996. J. Neurosci. 16:253).

In one embodiment, a linking group can be attached to a nucleomonomerand the transporting peptide can be covalently attached to the linker.In one embodiment, a linker can function as both an attachment site fora transporting peptide and can provide stability against nucleases.Examples of suitable linkers include substituted or unsubstituted C₁-C₂₀alkyl chains, C₂-C₂₀ alkenyl chains, C₂-C₂₀ alkynyl chains, peptides,and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers includebifunctional crosslinking agents such assulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smithet al. Biochem J 1991.276: 417-2).

In one embodiment, oligonucleotides of the invention are synthesized asmolecular conjugates which utilize receptor-mediated endocytoticmechanisms for delivering genes into cells (see, e.g., Bunnell et al.1992. Somatic Cell and Molecular Genetics. 18:559, and the referencescited therein).

Targeting Agents

The delivery of oligonucleotides can also be improved by targeting theoligonucleotides to a cellular receptor. The targeting moieties can beconjugated to the oligonucleotides or attached to a carrier group (i.e.,poly(L-lysine) or liposomes) linked to the oligonucleotides. This methodis well suited to cells that display specific receptor-mediatedendocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylatedproteins are internalized 20-fold more efficiently by cells expressingmannose 6-phosphate specific receptors than free oligonucleotides. Theoligonucleotides may also be coupled to a ligand for a cellular receptorusing a biodegradable linker. In another example, the delivery constructis mannosylated streptavidin which forms a tight complex withbiotinylated oligonucleotides. Mannosylated streptavidin was found toincrease 20-fold the internalization of biotinylated oligonucleotides.(Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

In addition specific ligands can be conjugated to the polylysinecomponent of polylysine-based delivery systems. For example,transferrin-polylysine, adenovirus-polylysine, and influenza virushemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugatesgreatly enhance receptor-mediated DNA delivery in eucaryotic cells.Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolarmacrophages has been employed to enhance the cellular uptake ofoligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

Because malignant cells have an increased need for essential nutrientssuch as folic acid and transferrin, these nutrients can be used totarget oligonucleotides to cancerous cells. For example, when folic acidis linked to poly(L-lysine) enhanced oligonucleotide uptake is seen inpromyelocytic leukaemia (HL-60) cells and human melanoma (M−14) cells.Ginobbi et al. 1997. Anticancer Res. 17:29. In another example,liposomes coated with maleylated bovine serum albumin, folic acid, orferric protoporphyrin IX, show enhanced cellular uptake ofoligonucleotides in murine macrophages, KB cells, and 2.2.15 humanhepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

Liposomes naturally accumulate in the liver, spleen, andreticuloendothelial system (so-called, passive targeting). By couplingliposomes to various ligands such as antibodies are protein A, they canbe actively targeted to specific cell populations. For example, proteinA-bearing liposomes may be pretreated with H-2K specific antibodieswhich are targeted to the mouse major histocompatibility complex-encodedH-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica etBiophysica Acta 1197:95-108).

Other in vitro and/or in vivo delivery of RNAi reagents are known in theart, and can to be used to deliver the subject RNAi constructs. See, forexample, U.S. patent application publications 20080152661, 20080112916,20080107694, 20080038296, 20070231392, 20060240093, 20060178327,20060008910, 20050265957, 20050064595, 20050042227, 20050037496,20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO2008/036825, WO04/065601, and AU2004206255B2, just to name a few (allincorporated by reference).

Administration

The optimal course of administration or delivery of the oligonucleotidesmay vary depending upon the desired result and/or on the subject to betreated. As used herein “administration” refers to contacting cells witholigonucleotides and can be performed in vitro or in vivo. The dosage ofoligonucleotides may be adjusted to optimally reduce expression of aprotein translated from a target nucleic acid molecule, e.g., asmeasured by a readout of RNA stability or by a therapeutic response,without undue experimentation.

For example, expression of the protein encoded by the nucleic acidtarget can be measured to determine whether or not the dosage regimenneeds to be adjusted accordingly. In addition, an increase or decreasein RNA or protein levels in a cell or produced by a cell can be measuredusing any art recognized technique. By determining whether transcriptionhas been decreased, the effectiveness of the oligonucleotide in inducingthe cleavage of a target RNA can be determined.

Any of the above-described oligonucleotide compositions can be usedalone or in conjunction with a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes appropriatesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, it can be used in thetherapeutic compositions. Supplementary active ingredients can also beincorporated into the compositions.

Oligonucleotides may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types.

With respect to in vivo applications, the formulations of the presentinvention can be administered to a patient in a variety of forms adaptedto deliver the construct to the eye. In preferred embodiments,parenteral administration is ocular. Ocular administration can beintravitreal, intracameral, subretinal, subconjunctival, or subtenon.

Pharmaceutical preparations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, thesuspension may also contain stabilizers. The oligonucleotides of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligonucleotides may be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included in the invention.

The chosen method of delivery will result in entry into cells. In someembodiments, preferred delivery methods include liposomes (10-400 nm),hydrogels, controlled-release polymers, and other pharmaceuticallyapplicable vehicles, and microinjection or electroporation (for ex vivotreatments).

The pharmaceutical preparations of the present invention may be preparedand formulated as emulsions. Emulsions are usually heterogeneous systemsof one liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. The emulsions of the present invention maycontain excipients such as emulsifiers, stabilizers, dyes, fats, oils,waxes, fatty acids, fatty alcohols, fatty esters, humectants,hydrophilic colloids, preservatives, and anti-oxidants may also bepresent in emulsions as needed. These excipients may be present as asolution in either the aqueous phase, oily phase or itself as a separatephase.

Examples of naturally occurring emulsifiers that may be used in emulsionformulations of the present invention include lanolin, beeswax,phosphatides, lecithin and acacia. Finely divided solids have also beenused as good emulsifiers especially in combination with surfactants andin viscous preparations. Examples of finely divided solids that may beused as emulsifiers include polar inorganic solids, such as heavy metalhydroxides, nonswelling clays such as bentonite, attapulgite, hectorite,kaolin, montrnorillonite, colloidal aluminum silicate and colloidalmagnesium aluminum silicate, pigments and nonpolar solids such as carbonor glyceryl tristearate.

Examples of preservatives that may be included in the emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Examples of antioxidants that may be included in the emulsionformulations include free radical scavengers such as tocopherols, alkylgallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

In one embodiment, the compositions of oligonucleotides are formulatedas microemulsions. A microemulsion is a system of water, oil andamphiphile which is a single optically isotropic and thermodynamicallystable liquid solution. Typically microemulsions are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a 4th component, generally an intermediatechain-length alcohol to form a transparent system.

Surfactants that may be used in the preparation of microemulsionsinclude, but are not limited to, ionic surfactants, non-ionicsurfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fattyacid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate(MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate(PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate(MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate(DA0750), alone or in combination with cosurfactants. The cosurfactant,usually a short-chain alcohol such as ethanol, 1-propanol, and1-butanol, serves to increase the interfacial fluidity by penetratinginto the surfactant film and consequently creating a disordered filmbecause of the void space generated among surfactant molecules.

Microemulsions may, however, be prepared without the use ofcosurfactants and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase may typically be, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase may include, but is not limited to, materials suchas Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fattyacid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both oil/water and water/oil) have been proposed toenhance the oral bioavailability of drugs.

Microemulsions offer improved drug solubilization, protection of drugfrom enzymatic hydrolysis, possible enhancement of drug absorption dueto surfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm.Sci., 1996, 85:138-143). Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides from thegastrointestinal tract, as well as improve the local cellular uptake ofoligonucleotides within the gastrointestinal tract, vagina, buccalcavity and other areas of administration.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular oligonucleotide and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved. When lipids are used to deliver theoligonucleotides, the amount of lipid compound that is administered canvary and generally depends upon the amount of oligonucleotide agentbeing administered. For example, the weight ratio of lipid compound tooligonucleotide agent is preferably from about 1:1 to about 15:1, with aweight ratio of about 5:1 to about 10:1 being more preferred. Generally,the amount of cationic lipid compound which is administered will varyfrom between about 0.1 milligram (mg) to about 1 gram (g). By way ofgeneral guidance, typically between about 0.1 mg and about 10 mg of theparticular oligonucleotide agent, and about 1 mg to about 100 mg of thelipid compositions, each per kilogram of patient body weight, isadministered, although higher and lower amounts can be used.

The agents of the invention are administered to subjects or contactedwith cells in a biologically compatible form suitable for pharmaceuticaladministration. By “biologically compatible form suitable foradministration” is meant that the oligonucleotide is administered in aform in which any toxic effects are outweighed by the therapeuticeffects of the oligonucleotide. In one embodiment, oligonucleotides canbe administered to subjects. Examples of subjects include mammals, e.g.,humans and other primates; cows, pigs, horses, and farming(agricultural) animals; dogs, cats, and other domesticated pets; mice,rats, and transgenic non-human animals.

Administration of an active amount of an oligonucleotide of the presentinvention is defined as an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example, an activeamount of an oligonucleotide may vary according to factors such as thetype of cell, the oligonucleotide used, and for in vivo uses the diseasestate, age, sex, and weight of the individual, and the ability of theoligonucleotide to elicit a desired response in the individual.Establishment of therapeutic levels of oligonucleotides within the cellis dependent upon the rates of uptake and efflux or degradation.Decreasing the degree of degradation prolongs the intracellularhalf-life of the oligonucleotide. Thus, chemically-modifiedoligonucleotides, e.g., with modification of the phosphate backbone, mayrequire different dosing.

The exact dosage of an oligonucleotide and number of doses administeredwill depend upon the data generated experimentally and in clinicaltrials. Several factors such as the desired effect, the deliveryvehicle, disease indication, and the route of administration, willaffect the dosage. Dosages can be readily determined by one of ordinaryskill in the art and formulated into the subject pharmaceuticalcompositions. Preferably, the duration of treatment will extend at leastthrough the course of the disease symptoms.

Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide may be repeatedlyadministered, e.g., several doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

Ocular administration of sd-rxRNAs, including intravitreal intravitreal,intracameral, subretinal, subconjunctival, and subtenon administration,can be optimized through testing of dosing regimens. In someembodiments, a single administration is sufficient. To further prolongthe effect of the administered sd-rxRNA, the sd-rxRNA can beadministered in a slow-release formulation or device, as would befamiliar to one of ordinary skill in the art. The hydrophobic nature ofsd-rxRNA compounds can enable use of a wide variety of polymers, some ofwhich are not compatible with conventional oligonucleotide delivery.

In other embodiments, the sd-rxRNA is administered multiple times. Insome instances it is administered daily, bi-weekly, weekly, every twoweeks, every three weeks, monthly, every two months, every three months,every four months, every five months, every six months or lessfrequently than every six months. In some instances, it is administeredmultiple times per day, week, month and/or year. For example, it can beadministered approximately every hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours 10 hours, 12 hours or morethan twelve hours. It can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more than 10 times per day.

Aspects of the invention relate to administering sd-rxRNA or rxRNA orimolecules to a subject. In some instances the subject is a patient andadministering the sd-rxRNA molecule involves administering the sd-rxRNAmolecule in a doctor's office.

FIG. 1 reveals uptake of sd-rxRNA 24 hours post intravitreal orsubretinal dosing. By 24 hours, sd-rxRNA has penetrated the entireretina. FIG. 2 shows a comparison of retinal uptake of sd-rxRNA andconventional RNAi compounds. While both are detected in the eyeimmediately after administration, after 48 hours, only the sd-rxRNA isdetected. FIG. 3 shows detection of sd-rxRNA throughout the retinafollowing intravitreal dosing, but not detection of conventional RNAicompounds. FIG. 4 shows that sd-rxRNA penetrates the retina to the outersegments of the photoreceptors.

In some instances, the effective amount of sd-rxRNA that is deliveredthrough ocular administration is at least approximately 0.05, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 ormore than 100 μg including any intermediate values.

sd-rxRNA molecules administered through methods described herein areeffectively targeted to all the cell types in the eye.

Physical methods of introducing nucleic acids include injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, electroporation of cell membranes in the presence of thenucleic acid or topical application of a composition comprising thenucleic acid to the eye. A viral construct packaged into a viralparticle would accomplish both efficient introduction of an expressionconstruct into the cell and transcription of nucleic acid encoded by theexpression construct. Other methods known in the art for introducingnucleic acids to cells may be used, such as lipid-mediated carriertransport, chemical-mediated transport, such as calcium phosphate, andthe like. Thus the nucleic acid may be introduced along with componentsthat perform one or more of the following activities: enhance nucleicacid uptake by the cell, inhibit annealing of single strands, stabilizethe single strands, or other-wise increase inhibition of the targetgene.

Assays of Oligonucleotide Stability

In some embodiments, the oligonucleotides of the invention arestabilized, i.e., substantially resistant to endonuclease andexonuclease degradation. An oligonucleotide is defined as beingsubstantially resistant to nucleases when it is at least about 3-foldmore resistant to attack by an endogenous cellular nuclease, and ishighly nuclease resistant when it is at least about 6-fold moreresistant than a corresponding oligonucleotide. This can be demonstratedby showing that the oligonucleotides of the invention are substantiallyresistant to nucleases using techniques which are known in the art.

One way in which substantial stability can be demonstrated is by showingthat the oligonucleotides of the invention function when delivered to acell, e.g., that they reduce transcription or translation of targetnucleic acid molecules, e.g., by measuring protein levels or bymeasuring cleavage of mRNA. Assays which measure the stability of targetRNA can be performed at about 24 hours post-transfection (e.g., usingNorthern blot techniques, RNase Protection Assays, or QC-PCR assays asknown in the art). Alternatively, levels of the target protein can bemeasured. Preferably, in addition to testing the RNA or protein levelsof interest, the RNA or protein levels of a control, non-targeted genewill be measured (e.g., actin, or preferably a control with sequencesimilarity to the target) as a specificity control. RNA or proteinmeasurements can be made using any art-recognized technique. Preferably,measurements will be made beginning at about 16-24 hours posttransfection. (M. Y. Chiang, et al. 1991. J Biol Chem. 266:18162-71; T.Fisher, et al. 1993. Nucleic Acids Research. 21 3857).

The ability of an oligonucleotide composition of the invention toinhibit protein synthesis can be measured using techniques which areknown in the art, for example, by detecting an inhibition in genetranscription or protein synthesis. For example, Nuclease Si mapping canbe performed. In another example, Northern blot analysis can be used tomeasure the presence of RNA encoding a particular protein. For example,total RNA can be prepared over a cesium chloride cushion (see, e.g.,Ausebel et al., 1987. Current Protocols in Molecular Biology (Greene &Wiley, New York)). Northern blots can then be made using the RNA andprobed (see, e.g., Id.). In another example, the level of the specificmRNA produced by the target protein can be measured, e.g., using PCR. Inyet another example, Western blots can be used to measure the amount oftarget protein present. In still another embodiment, a phenotypeinfluenced by the amount of the protein can be detected. Techniques forperforming Western blots are well known in the art, see, e.g., Chen etal. J. Biol. Chem. 271:28259.

In another example, the promoter sequence of a target gene can be linkedto a reporter gene and reporter gene transcription (e.g., as describedin more detail below) can be monitored. Alternatively, oligonucleotidecompositions that do not target a promoter can be identified by fusing aportion of the target nucleic acid molecule with a reporter gene so thatthe reporter gene is transcribed. By monitoring a change in theexpression of the reporter gene in the presence of the oligonucleotidecomposition, it is possible to determine the effectiveness of theoligonucleotide composition in inhibiting the expression of the reportergene. For example, in one embodiment, an effective oligonucleotidecomposition will reduce the expression of the reporter gene.

A “reporter gene” is a nucleic acid that expresses a detectable geneproduct, which may be RNA or protein. Detection of mRNA expression maybe accomplished by Northern blotting and detection of protein may beaccomplished by staining with antibodies specific to the protein.Preferred reporter genes produce a readily detectable product. Areporter gene may be operably linked with a regulatory DNA sequence suchthat detection of the reporter gene product provides a measure of thetranscriptional activity of the regulatory sequence. In preferredembodiments, the gene product of the reporter gene is detected by anintrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detectable signal based on color, fluorescence, orluminescence. Examples of reporter genes include, but are not limitedto, those coding for chloramphenicol acetyl transferase (CAT),luciferase, beta-galactosidase, and alkaline phosphatase.

One skilled in the art would readily recognize numerous reporter genessuitable for use in the present invention. These include, but are notlimited to, chloramphenicol acetyltransferase (CAT), luciferase, humangrowth hormone (hGH), and beta-galactosidase. Examples of such reportergenes can be found in F. A. Ausubel et al., Eds., Current Protocols inMolecular Biology, John Wiley & Sons, New York, (1989). Any gene thatencodes a detectable product, e.g., any product having detectableenzymatic activity or against which a specific antibody can be raised,can be used as a reporter gene in the present methods.

One reporter gene system is the firefly luciferase reporter system.(Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408incorporated herein by reference). The luciferase assay is fast andsensitive. In this assay, a lysate of the test cell is prepared andcombined with ATP and the substrate luciferin. The encoded enzymeluciferase catalyzes a rapid, ATP dependent oxidation of the substrateto generate a light-emitting product. The total light output is measuredand is proportional to the amount of luciferase present over a widerange of enzyme concentrations.

CAT is another frequently used reporter gene system; a major advantageof this system is that it has been an extensively validated and iswidely accepted as a measure of promoter activity. (Gorman C. M.,Moffat, L. F., and Howard, B. H.1982. Mol. Cell. Biol., 2:1044-1051). Inthis system, test cells are transfected with CAT expression vectors andincubated with the candidate substance within 2-3 days of the initialtransfection. Thereafter, cell extracts are prepared. The extracts areincubated with acetyl CoA and radioactive chloramphenicol. Following theincubation, acetylated chloramphenicol is separated from nonacetylatedform by thin layer chromatography. In this assay, the degree ofacetylation reflects the CAT gene activity with the particular promoter.

Another suitable reporter gene system is based on immunologic detectionof hGH. This system is also quick and easy to use. (Selden, R.,Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986),Mol. Cell, Biol., 6:3173-3179 incorporated herein by reference). The hGHsystem is advantageous in that the expressed hGH polypeptide is assayedin the media, rather than in a cell extract. Thus, this system does notrequire the destruction of the test cells. It will be appreciated thatthe principle of this reporter gene system is not limited to hGH butrather adapted for use with any polypeptide for which an antibody ofacceptable specificity is available or can be prepared.

In one embodiment, nuclease stability of a double-strandedoligonucleotide of the invention is measured and compared to a control,e.g., an RNAi molecule typically used in the art (e.g., a duplexoligonucleotide of less than 25 nucleotides in length and comprising 2nucleotide base overhangs) or an unmodified RNA duplex with blunt ends.

The target RNA cleavage reaction achieved using the siRNAs of theinvention is highly sequence specific. Sequence identity may determinedby sequence comparison and alignment algorithms known in the art. Todetermine the percent identity of two nucleic acid sequences (or of twoamino acid sequences), the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence or secondsequence for optimal alignment). A preferred, non-limiting example of alocal alignment algorithm utilized for the comparison of sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. Additionally, numerous commercial entities, such asDharmacon, and Invitrogen provide access to algorithms on their website.The Whitehead Institute also offers a free siRNA Selection Program.Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target gene is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetgene transcript. Examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference.

Therapeutic Use

By inhibiting the expression of a gene, the oligonucleotide compositionsof the present invention can be used to treat any disease involving theexpression of a protein. Examples of diseases that can be treated byoligonucleotide compositions, just to illustrate, include: cancer,retinopathies, autoimmune diseases, inflammatory diseases (i.e., ICAM-1related disorders, Psoriasis, Ulcerative Colitus, Crohn's disease),viral diseases (i.e., HIV, Hepatitis C), miRNA disorders, andcardiovascular diseases.

As discussed above, sd-rxRNA molecules administered by methods describedherein are effectively targeted to all the cell types in the eye.

Aspects of the invention relate to targeting sd-rxRNA to various celltypes in the eye, including, but not limited to, cells located in theganglion cell layer (GCL), the inner plexiform layer inner (IPL), theinner nuclear layer (INL), the outer plexiform layer (OPL), outernuclear layer (ONL), outer segments (OS) of rods and cones, the retinalpigmented epithelium (RPE), the inner segments (IS) of rods and cones,the epithelium of the conjunctiva, the iris, the ciliary body, thecorneum, and epithelium of ocular sebaceous glands.

The sd-rxRNA that is targeted to the eye may, in some instances targetan eye-specific gene or a gene that is expressed at higher levels in theeye than in other tissues. As one of ordinary skill in the art wouldappreciate, publicly accessible databases can be used to identify genesthat have eye-specific expression or increased expression in the eyerelative to other tissues. Several non-limiting examples of suchdatabases include TISGED (Tissue-Specific Genes Database) and the TiGERdatabase for tissue-specific gene expression and regulation. In otherembodiments, the sd-rxRNA does not target an eye-specific gene. In otherembodiments, the gene that is targeted does not have eye-specificexpression or increased expression in the eye.

In some instances, an sd-rxRNA that is targeted to the eye is used toameliorate at least one symptom of a condition or disorder associatedwith the eye. Several non-limiting examples of conditions or disordersassociated with the eye include: vascular leakage/neovascularization(e.g., angiographic cystoid macular edema, macular edema secondary toretinal vein occlusion (RVO), glaucoma or neovascular glaucoma (NVG),retinopathy of prematurity (ROP); fibroproliferative diseases (e.g.,proliferative vitreoretinopathy (PVR), epiretinalmembranes/vitreomacular adhesions; age-related macular degeneration(AMD) (e.g., choroidal neovascularization (wet AMD), geographic atrophy(advanced dry AMD), early-to-intermediate dry AMD); diabetic retinopathy(e.g., nonproliferative diabetic retinopathy (NPDR), diabetic macularedema (DME), proliferative diabetic retinopathy (PDR); retinaldegenerative diseases (and related diseases); retinal vascular occlusivediseases (e.g., retinal vein occlusion, retinal artery occlusion) andother retinal diseases; retinal detachment; inflammatory diseases suchas uveitis (including panuveitis) or choroiditis (including multifocalchoroiditis) of unknown cause (idiopathic) or associated with a systemic(e.g., autoimmune) disease; episcleritis or scleritis; Birdshotretinochoroidopathy; vascular diseases (retinal ischemia, retinalvasculitis, choroidal vascular insufficiency, choroidal thrombosis);neovascularization of the optic nerve; optic neuritis; blepharitis;keratitis; rubeosis iritis; Fuchs' heterochromic iridocyclitis; chronicuveitis or anterior uveitis; conjunctivitis; allergic conjunctivitis(including seasonal or perennial, vernal, atopic, and giant papillary);keratoconjunctivitis sicca (dry eye syndrome); iridocyclitis; iritis;scleritis; episcleritis; corneal edema; scleral disease; ocularcicatrcial pemphigoid; pars planitis; Posner Schlossman syndrome;Behcet's disease; Vogt-Koyanagi-Harada syndrome; hypersensitivityreactions; conjunctival edema; conjunctival venous congestion;periorbital cellulitis; acute dacryocystitis; non-specific vasculitis;sarcoidosis; keratoconjunctivitis sicca, a condition also known asdry-eye, keratitis sicca, sicca syndrome, xeropthalmia, and dry eyesyndrome (DES), which can arise from decreased tear production and/orincreased tear film evaporation due to abnormal tear composition; adisorder associated with the autoimmune diseases rheumatoid arthritis,lupus erythematosus, diabetes mellitus, and Sjogren's syndrome. In someembodiments, sd-rxRNA is administered as a method of wound healing.Non-limiting examples of conditions or disorders associated with the eyeare incorporated by reference from US Patent Publication 20100010082 andU.S. Pat. No. 6,331,313.

Neovascularization/Vascular Leakage

Aspects of the invention relate to treating diseases and conditionsassociated with neovascularization and/or vascular leakage. Of theseconditions, wet AMD and DME are most prevalent, PDR and macular edemasecondary to RVO are of lower prevalence, and rare neovascularconditions include ROP and neovascular glaucoma. Vascular leakage isconsidered to be the driving force behind DME, while both vascularleakage and neovascularization drive PDR. Oligonucleotide compositionsof the present invention can be selected based on the etiology of aparticular disease or condition. For example, a composition comprisingan anti-angiogenic oligonucleotide affecting vascular permeability maybe chosen to treat DME, while one affecting proliferation may be chosento treat PDR. Alternatively, oligonucleotide compositions may comprise acombination of anti-angiogenic agents, for example, an sd-rxRNA thatinhibits function of a target that affects vascular permeability and ansd-rxRNA that inhibits function of a target that affects proliferation,such that both etiological aspects of the condition are targeted.

In certain embodiments, the sd-rxRNA is used to treat neovascularizationand/or vascular permeability. In some embodiments, the sd-rxRNA targetsVascular Endothelial Growth Factor (VEGF), an inhibitor of vascularpermeability. VEGF is a canonical and clinically validated target fortreatment of wet AMD and approval is expected for DME and RVO-associatedME. VEGF proteins are growth factors that bind to tyrosine kinasereceptors and are implicated in multiple disorders such as cancer,age-related macular degeneration, rheumatoid arthritis and diabeticretinopathy. Members of this protein family include VEGF-A, VEGF-B,VEGF-C and VEGF-D. Representative Genbank accession numbers providingDNA and protein sequence information for human VEGF proteins areNM_(—)001171623.1 (VEGF-A), U43368 (VEGF-B), X94216 (VEGF-C), and D89630(VEGF-D).

Aspects of the invention relate to rxRNAori directed against VEGF. Asdescribed in the Examples section, over 100 optimal rxRNA ori sequencesfor VEGF were identified herein (Tables 2 and 9). An rxRNAori can bedirected against a sequence comprising at least 12 contiguousnucleotides of a sequence within Table 2 or 9. For example, an rxRNAorican be directed against a sequence comprising 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or contiguous nucleotides of a sequencewithin Table 2 or 9. In some embodiments, an rxRNAori is directedagainst a sequence comprising at least 12 contiguous nucleotides of SEQID NO:13 (AUCACCAUCGACAGAACAGUCCUUA) or SEQ ID NO: 28(CCAUGCAGAUUAUGCGGAUCAAACA). The sense strand of the rxRNAori moleculecan comprise at least 12 contiguous nucleotides of a sequence selectedfrom the sequences presented in Table 2. In some embodiments, the sensestrand of the rxRNAori comprises at least 12 contiguous nucleotides ofthe sequence of SEQ ID NO:13 or SEQ ID NO: 28. The antisense strand ofthe rxRNAori can be complementary to at least 12 contiguous nucleotidesof a sequence selected from the sequences within Table 2. In someembodiments, the antisense strand of the rxRNAori comprises at least 12contiguous nucleotides of SEQ ID NO:1377 (UAAGGACUGUUCUGUCGAUGGUGAU) orSEQ ID NO:1378 (UGUUUGAUCCGCAUAAUCUGCAUGG).

Non-limiting examples of an rxRNAori directed against VEGF include anrxRNAori comprising a sense strand that comprises the sequence of SEQ IDNO:13 and an antisense strand that comprises the sequence of SEQ IDNO:1377 or an rxRNAori comprising a sense strand that comprises thesequence of SEQ ID NO:28 and an antisense strand that comprises thesequence of SEQ ID NO:1378. It should be appreciated that a variety ofmodifications patterns are compatible with rxRNAori. Aspects of theinvention encompass rxRNAori directed against VEGF, wherein the rxRNAoriis modified or unmodified. In some embodiments, the rxRNAori isadiminstered to the eye.

Ori sequences can also be converted to sd-rxRNA molecules to target VEGFin the eye. It should be appreciated that the disclosed ori sequencesrepresent non-limiting examples of sequences within VEGF for sd-rxRNAdevelopment. Variations in length and modifications of these sequences,as well as other sequences within VEGF are also compatible withdevelopment of sd-rxRNA molecules. An sd-rxRNA can be directed against asequence selected from the sequences within Table 2 or 9. For example,an sd-rxRNA can be directed against a sequence comprising at least 12contiguous nucleotides of a sequence selected from the sequences withinTable 2 or 9. In some embodiments, an sd-rxRNA can be directed against asequence comprising 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24or 25 contiguous nucleotides of a sequence selected from the sequenceswithin Table 2 or 9.

In some embodiments, an sd-rxRNA directed against VEGF comprises atleast 12 nucleotides of a sequence selected from the sequences withinTable 8. In some embodiments, the sense strand of the sd-rxRNA comprisesat least 12 contiguous nucleotides of the sequence of SEQ ID NO:1317(AGAACAGUCCUUA) or SEQ ID NO:1357 (UGCGGAUCAAACA) and/or the antisensestrand of the sd-rxRNA comprises at least 12 contiguous nucleotides ofthe sequence of SEQ ID NO:1318 (UAAGGACUGUUCUGUCGAU) or SEQ ID NO:1358(UGUUUGAUCCGCAUAAUCU). In certain embodiments, an sd-rxRNA directedagainst VEGF includes a sense strand comprising SEQ ID NO:1317 and anantisense strand comprising SEQ ID NO:1318. Various chemicalmodification patterns are compatible with sd-rxRNA. Non-limitingexamples of modified forms of SEQ ID NO:1317 and SEQ ID NO:1318 arerepresented by SEQ ID NOs 1379 (A. G. A. A.mC. A. G.mU.mC.mC.mU.mU.A.Chl) and 1380 (P.mU. A. A. G. G. A.fC.fU. G.fU.fU.fC.fU* G*fU*fC* G*A* U), respectively.

In certain embodiments, an sd-rxRNA directed against VEGF includes asense strand comprising SEQ ID NO:1357 and an antisense strandcomprising SEQ ID NO:1358. Non-limiting examples of modified forms ofSEQ ID NO:1357 and SEQ ID NO:1358 are represented by SEQ ID NOs 1397(mU. G.mC. G. G. A.mU.mC. A. A. A.mC. A.Chl) and 1398 (P.mU. G.fU.fU.fU.G. A.fU.fC.fC. G.fC. A*fU* A* A*fU*fC* U), respectively. In certainembodiments, the sd-rxRNA comprises SEQ ID NOs 1397 and 1398. It shouldbe appreciated that other modifications patterns of sd-rxRNAs disclosedherein are also compatible with aspects of the invention.

Described herein are also sd-rxRNAs directed against genes that encodefor proteins other than VEGF. Non-limiting examples of such sd-rxRNAsare provided in Tables 3-7. In some embodiments, an sd-rxRNA comprisesat least 12 contiguous nucleotides of a sequence selected from thesequences within Tables 3-7.

In some embodiments, the sd-rxRNA is directed against CTGF. Non-limitingexamples of sd-rxRNAs directed against CTGF are provided in Table 5. Insome embodiments, the sense strand of an sd-rxRNA directed against CTGFcomprises at least 12 contiguous nucleotides of the sequence of SEQ IDNO:1422 (GCACCUUUCUAGA) and an antisense strand of an sd-rxRNA directedagainst CTGF comprises at least 12 contiguous nucleotides of thesequence of SEQ ID NO:1423 (UCUAGAAAGGUGCAAACAU). Non-limiting examplesof modified forms of SEQ ID NOs 1422 and 1423 are represented by SEQ IDNOs:947 (G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA.TEG-Chl) and 948(P.mU.fC.fU. A. G.mA. A.mA. G. G.fU. G.mC* A* A* A*mC* A* U.),respectively. In some embodiments, the sense strand of an sd-rxRNAdirected against CTGF comprises at least 12 contiguous nucleotides ofthe sequence of SEQ ID NO:1424 (UUGCACCUUUCUAA) and an antisense strandof an sd-rxRNA directed against CTGF comprises at least 12 contiguousnucleotides of the sequence of SEQ ID NO:1425 (UUAGAAAGGUGCAAACAAGG).Non-limiting examples of modified forms of SEQ ID Nos 1424 and 1425 andrepresented by SEQ ID NOs 963 (mU.mU. G.mC.A.mC.mC.mU.mU.mU.mC.mU*mA*mA.TEG-Chl) and 964 (P.mU.fU. A. G. A.mA. A.G. G.fU. G.fC.mA.mA*mA*fC*mA*mA*mG* G.).

In some embodiments, the sense strand of the sd-rxRNA directed againstCTGF comprises at least 12 contiguous nucleotides of the sequence of SEQID NO:947 or SEQ ID NO:963. In certain embodiments, the sd-rxRNAdirected against CTGF includes a sense strand comprising the sequence ofSEQ ID NO:963 and an antisense strand comprising the sequence of SEQ IDNO:964. In other embodiments, the sd-rxRNA directed against CTGFincludes a sense strand comprising the sequence of SEQ ID NO:947 and anantisense strand comprising the sequence of SEQ ID NO:948.

sd-rxRNA can be hydrophobically modified. For example, the sd-rxRNA canbe linked to one or more hydrophobic conjugates. In some embodiments,the sd-rxRNA includes at least one 5-methyl C or U modifications.

Aspects of the invention relate to compositions comprising rxRNAoriand/or sd-rxRNA nucleic acids described herein. A composition cancomprise one or more rxRNAori and/or sd-rxRNA. In some embodiments, acomposition comprises multiple different rxRNAoris that are directed togenes encoding for different proteins and/or multiple differentsd-rxRNAs that are directed to genes encoding for different proteins. Insome embodiments, a composition comprises sd-rxRNA directed to VEGF aswell as sd-rxRNA directed against another gene such as a gene encodingfor CTGF or PTGS2 (COX-2). In some embodiments, one or more sd-rxRNAtargets IGTA5, ANG2, CTGF, COX-2, complement factors 3 or 5, or acombination thereof.

In some embodiments, the sd-rxRNA targets Connective tissue growthfactor (CTGF), also known as Hypertrophic chondrocyte-specific protein24. CTGF is a secreted heparin-binding protein that has been implicatedin wound healing and scleroderma. Connective tissue growth factor isactive in many cell types including fibroblasts, myofibroblasts,endothelial and epithelial cells. Representative Genbank accessionnumber providing DNA and protein sequence information for human CTGF areNM_(—)001901.2 and M92934.

In some embodiments, the sd-rxRNA targets Osteopontin (OPN), also knownas Secreted phosphoprotein 1 (SPP1), Bone Sinaloprotein 1 (BSP-1), andearly T-lymphocyte activation (ETA-1). SPP1 is a secreted glycoproteinprotein that binds to hydroxyapatite. OPN has been implicated in avariety of biological processes including bone remodeling, immunefunctions, chemotaxis, cell activation and apoptosis. Osteopontin isproduced by a variety of cell types including fibroblasts,preosteoblasts, osteoblasts, osteocytes, odontoblasts, bone marrowcells, hypertrophic chondrocytes, dendritic cells, macrophages, smoothmuscle, skeletal muscle myoblasts, endothelial cells, and extraosseous(non-bone) cells in the inner ear, brain, kidney, deciduum, andplacenta. Representative Genbank accession number providing DNA andprotein sequence information for human Osteopontin are NM_(—)000582.2and X13694.

In some embodiments, the sd-rxRNA targets Transforming growth factor β(TGFβ) proteins, for which three isoforms exist in mammals (TGFβ1,TGFβ2, TGFβ3). TGFβ proteins are secreted proteins belonging to asuperfamily of growth factors involved in the regulation of manycellular processes including proliferation, migration, apoptosis,adhesion, differentiation, inflammation, immuno-suppression andexpression of extracellular proteins. These proteins are produced by awide range of cell types including epithelial, endothelial,hematopoietic, neuronal, and connective tissue cells. RepresentativeGenbank accession numbers providing DNA and protein sequence informationfor human TGFβ1, TGFβ2 and TGFβ3 are BT007245, BC096235, and X14149,respectively. Within the TGFβ family, TGFβ1 and TGFβ2 but not TGFβ3represent suitable targets. In some embodiments, the sd-rxRNA targetsCyclooxygenase-2 (COX-2), also called Prostaglandin G/H synthase 2(PTGS2). COX-2 is involved in lipid metabolism and biosynthesis ofprostanoids and is implicated in inflammatory disorders such asrheumatoid arthritis. A representative Genbank accession numberproviding DNA and protein sequence information for human COX-2 isAY462100.

In other embodiments, the sd-rxRNA targets HIF-1α, a component of theHIF-1 transcription factor. HIF-1α is a key regulator of the cellularresponse to hypoxia, acting upstream of VEGF-dependent andVEGF-independent pro-angiogenic pathways and pro-fibrotic pathways.HIF-1α inhibitors are effective in laser CNV and OIR models. Arepresentative Genbank accession number providing DNA and proteinsequence information for human HIF1α is U22431.

In some embodiments, the sd-rxRNA targets mTOR. mTOR is aserine/threonine kinase component of the PI3K/Akt/mTOR pathway, and is aregulator or cell growth, proliferation, survival, transcription andtranslation. mTOR inhibitors have both anti-angiogenic (effective inlaser CNV and OIR models) and anti-fibrotic activity. Rapamycin andother mTOR inhibitors are being used in clinical trials for AMD and DME.A representative Genbank accession number providing DNA and proteinsequence information for human mTOR is L34075.

In some embodiments, the sd-rxRNA targets SDF-1 (stromal derivedfactor-1), which is a soluble factor that stimulates homing ofhematopoietic stem cells and endothelial progenitor cells to tissues.SDF-1 acts synergistically with VEGF to drive pathologicneovascularization, and inhibition of SDF-1 signaling suppressesneovascularization in OIR, laser CNV, and VEGF-induced rodent models.

In certain embodiments, the sd-rxRNA targets PDGF-B (platelet-derivedgrowth factor B). Retinal overexpression of PDGF-B in transgenic miceleads to fibrovascular proliferation, and inhibition of PDGF-B signalingenhances efficacy of anti-VEGF treatment in laser CNV model. Dualinhibition of PDGF-B and VEGF can promote regression of NV.Representative Genbank accession numbers providing DNA and proteinsequence information for human PDGF genes and proteins include X03795(PDGFA), X02811 (PDGFB), AF091434 (PDGFC), AB033832 (PDGFD).

In some embodiments, the sd-rxRNA targets TIE1 (tyrosine kinase withimmunoglobulin-like and EGF-like domains).

In other embodiments, the sd-rxRNA targets VEGFR1 (vascular endothelialgrowth factor receptor 1), also referred to as FLT1 (fms-relatedtyrosine kinase 1). This gene encodes a member of the vascularendothelial growth factor receptor (VEGFR) family. VEGFR family membersare receptor tyrosine kinases (RTKs) which contain an extracellularligand-binding region with seven immunoglobulin (Ig)-like domains, atransmembrane segment, and a tyrosine kinase (TK) domain within thecytoplasmic domain. This protein binds to VEGFR-A, VEGFR-B and placentalgrowth factor and plays an important role in angiogenesis andvasculogenesis. Representative Genbank accession numbers providing DNAand protein sequence information for human VEGFR1 genes and proteinsinclude NM_(—)001159920, NP_(—)001153392, NM_(—)001160030,NP_(—)001153502, NM_(—)001160031, NP_(—)001153503, NM_(—)002019, andNP_(—)002010.

In certain embodiments, the sd-rxRNA targets VEGFR2 (vascularendothelial growth factor receptor 2), also referred to as KDR (kinaseinsert domain receptor). This receptor, known as kinase insert domainreceptor, is a type III receptor tyrosine kinase. It functions as themain mediator of VEGF-induced endothelial proliferation, survival,migration, tubular morphogenesis and sprouting. The signaling andtrafficking of this receptor are regulated by multiple factors,including Rab GTPase, P2Y purine nucleotide receptor, integrinalphaVbeta3, T-cell protein tyrosine phosphatase, etc. RepresentativeGenbank accession numbers providing DNA and protein sequence informationfor human VEGFR2 genes and proteins include NM_(—)002253 andNP_(—)002244. In some embodiments, treatment of neovascularizationand/or vascular leakage may include the use of a combination ofsd-rxRNAs, each sd-rxRNA targeting a different gene. For example, ansd-rRNA targeting VEGF and an sd-rxRNA targeting HIF-1α can be used. Asanother example, an sd-rRNA targeting mTOR and an sd-rRNA targetingSDF-1 can be used. As yet another example, an sd-rRNA targeting VEGF, ansd-rRNA targeting mTOR, and an sd-rRNA targeting PDGF-B can be used.

Wet AMD (Choroidal Neovascularization (CNV))

Aspects of the invention relate to treating choroidal vascularization,the fastest progressing form of AMD (−1 million cases in the U.S.),which results from inappropriate growth of new blood vessels from thechoroid into the subretinal space and leakage of fluid from thesevessels. If untreated, 75% of patients will progress to legal blindnesswithin three years. Intravitreal anti-VEGF agents can rapidly improvevision by inhibiting CNV lesion growth and vascular leakage from CNVlesions; however, existing anti-VEGFs may not cause regression ofexisting lesions in most patients.

In certain embodiments, the sd-rxRNA is used to treat CNV. In someembodiments, the sd-rxRNA targets VEGF. In other embodiments, thesd-rxRNA targets HIF-1α, mTOR, PDGF-B, SDF-1, IGTA5, ANG2, CTGF, COX-2,or complement factors 3 or 5. In some embodiments, treatment of CNVincludes the use of a combination of sd-rxRNAs, each sd-rxRNA targetinga different gene.

Diabetic Macular Edema (DME)

DME results from vascular leakage from retinal vessels leading tovision-threatening buildup of fluid in the macula, occurring in ˜2-5% ofdiabetic patients. The current standard of care is focal or grid laserphotocoagulation. Intravitreal anti-VEGF agents and corticosteroids havebeen shown to be effective, but are not yet approved.

In certain embodiments, the sd-rxRNA is used to treat DMA. In someembodiments, the sd-rxRNA targets VEGF. In other embodiments, thesd-rxRNA targets HIF-1α, mTOR, PDGF-B, SDF-1, IGTA5, ANG2, CTGF, COX-2,or complement factors 3 or 5. In some embodiments, treatment of DMEincludes the use of a combination of sd-rxRNAs, each sd-rxRNA targetinga different gene.

Proliferative Diabetic Retinopathy (PDR)

PDR is associated with chronic retinal ischemia. Retinalneovascularization occurs secondary to retinal ischemia and can lead tovitreous hemorrhage, fibrovascular proliferation, and traction retinaldetachment.

In certain embodiments, the sd-rxRNA is used to treat PDR. In someembodiments, the sd-rxRNA targets VEGF. In other embodiments, thesd-rxRNA targets HIF-1α, mTOR, PDGF-B, SDF-1, IGTA5, ANG2, CTGF, COX-2,or complement factors 3 or 5. In some embodiments, treatment of PDRincludes the use of a combination of sd-rxRNAs, each sd-rxRNA targetinga different gene.

Macular Edema Secondary to RVO

RVO can occur in ischemic and non-ischemic forms. Ischemic RVO can leadto several vision threatening complications, including macular edema,retinal ischemia, and neovascularization. Non-ischemic RVO has a morefavorable prognosis and the most common vision-threatening complicationis macular edema.

In certain embodiments, the sd-rxRNA is used to treat macular edemasecondary to RVO. In some embodiments, the sd-rxRNA targets VEGF. Inother embodiments, the sd-rxRNA targets HIF-1α, mTOR, PDGF-B, SDF-1,IGTA5, ANG2, CTGF, COX-2, or complement factors 3 or 5. In someembodiments, treatment of macular edema secondary to RVO includes theuse of a combination of sd-rxRNAs, each sd-rxRNA targeting a differentgene.

Iris Neovascularization/Neovascular Glaucoma (NVG)

NVG is a rare disorder that develops in eyes suffering from severe,chronic ocular ischemia. The most common causes are advanced PDR orischemic CRVO. his neovascularization occurs due to ischemia, andeventually obstructs trabecular meshwork leading to a severe secondaryglaucoma.

In certain embodiments, the sd-rxRNA is used to treat irisneovascularization and/or NVG. In some embodiments, the sd-rxRNA targetsVEGF. In other embodiments, the sd-rxRNA targets HIF-1α, mTOR, PDGF-B,SDF-1, IGTA5, ANG2, CTGF, COX-2, or complement factors 3 or 5. In someembodiments, treatment of iris neovascularization and/or NVG includesthe use of a combination of sd-rxRNAs, each sd-rxRNA targeting adifferent gene.

Proliferative Retinal Diseases

Proliferative retinal diseases include proliferative vitreoretinopathy,proliferative diabetic retinopathy (PDR), epiretinal membranes(transparent layers of cells that can grow over the surface of themacula, causing retinal traction), and wet AMD.

In certain embodiment, the sd-rxRNA is used to treat proliferativeretinal diseases. In some embodiments, the sd-rxRNA targets TGFβ, whilein other embodiments, the sd-rxRNA targets CTGF. In still otherembodiments, multiple sd-rxRNAs target PDGFRα, mTOR, IGTA5, or acombination thereof. In yet other embodiments, multiple sd-rxRNAstargets TGFβ and at least one of CTGF, PDGFRα, mTOR, IGTA5, or acombination thereof. In further embodiments, multiple sd-rxRNAs targetCTGF and at least one of TGFβ, PDGFRα, mTOR, IGTA5, or a combinationthereof. In certain embodiments, treatment of proliferative retinaldiseases includes the use of a combination of sd-rxRNAs, each sd-rxRNAtargeting a different gene.

Dry AMD

In certain embodiments, the sd-rxRNA is used to treat dry AMD, includinggeographic atrophy (GA) (a form of advanced AMD that progresses moreslowly than wet AMD) and early-to-intermediate dry AMD (early stages ofdry AMD that precedes GA or CNV). In some embodiments, the sd-rxRNAtargets Alu transcription. In other embodiments, the sd-rxRNA targetstranscription factors or other molecules that inhibit or regulateexpression of DICER (an endoribonuclease in the RNase III family thatcleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA) into shortdouble-stranded RNA fragments called small interfering RNA (siRNA) about20-25 nucleotides long).

Cystoid Macular Edema

Cystoid macular edema is an accumulation of intraretinal fluid inerofoveal cysts following surgery. In certain embodiments, the sd-rxRNAis used to treat cystoid macular edema. In some embodiments, thesd-rxRNA targets COX-2 (cyclooxygenase-2) enzyme.

Retinitis Pigmentosa

Retinitis pigmentosa is an inherited retinal degenerative disease causedby mutations in several known genes. In certain embodiments, thesd-rxRNA is used to treat retinitis pigmentosa. In some embodiments, thesd-rxRNA targets NADPH oxidase.

Glaucoma

Glaucoma is a slowly progressive disease characterized by degenerationof the optic nerve. There is an initial vision loss in the peripherywith central vision loss at advanced stages of the disease. The bestunderstood risk factor for glaucoma-related vision loss is intraocularpressure (IOP). Trabeculectomy is a surgical procedure designed tocreate a channel or bleb though the sclera to allow excess fluid todrain from the anterior of the eye, leading to reduced IOP. The mostcommon cause of trabeculectomy failure is blockage of the bleb by scartissue.

In certain embodiments, the sd-rxRNA is used to prevent formation ofscar tissue resulting from a trabeculectomy. In some embodiments, thesd-rxRNA targets CTGF, while in other embodiments, the sd-rxRNA targetsTGFβ. In still other embodiments, multiple sd-rxRNAs target both CTGFand TGFβ. In some embodiments, scar tissue formation is prevented by theuse of a combination of sd-rxRNAs, one targeting CTGF and one targetingTGFβ.

Uveitis

Uveitis is a broad group of disorders characterized by inflammation ofthe middle layer of the eye, called the uvea, which is composed of thechoroid, ciliary body, and iris. The disorders are categorizedanatomically as anterior, intermediate, posterior, or panuveitis, andare categorized pathologically as infectious or non-infectious.

In certain embodiments, the sd-rxRNA is used to treat uveitis. In someembodiments, the sd-rxRNA targets a cytokine, for example TNFα. In otherembodiments, the sd-rxRNA targets IL-1, IL-6, IL-15, IL-17, IL-2R, orCTLA-4. In still other embodiments, the sd-rxRNA targets adhesionmolecules, including VLA-4, VCAM-1, LFA-1, ICAM-1, CD44, or osteopontin.In yet another embodiment, the sd-rxRNA targets at least one of TNFα,IL-1, IL-6, IL-15, IL-17, IL-2R, CTLA-4, VLA-4, VCAM-1, LFA-1, ICAM-1,CD44, and osteopontin. In some embodiments, scar tissue formation isprevented by the use of a combination of sd-rxRNAs, each targeting adifferent gene.

Retinoblastoma (Rb)

Retinoblastoma is a rapidly developing cancer in the cells of retina. Incertain embodiments, the sd-rxRNA is used to treat retinoblastoma. Insome embodiments, the sd-rxRNA targets HMGA2, a nuclear protein thoughtto have a role in neoplastic transformation.

In certain embodiments, sd-rxRNAs of the present invention can be usedfor multi-gene silencing. In some embodiments, a combination ofsd-rxRNAs is used to target multiple, different genes. For example, whenused for the treatment of a neovascular disorder, a sd-rxRNA targetingVEGF can be used together with a sd-rxRNA targeting HIF-1α. As anotherexample, when used for the treatment of uveitis, a sd-rxRNA targetingTNFα, a sd-rxRNA targeting VCAM-1, and a sd-rxRNA targeting IL-2R can beused in combination.

In some embodiments, multiple sd-rxRNAs can be used to target VEGF,IGTA5, ANG2, CTGF, COX-2, complement factor 3, complement factor 5,HIF-1α, mTOR, SDF-1, PDGF-β, Alu, NADPH oxidase, TGF-β, IL-1, IL-6,IL-15, IL-17, IL-2R, CTLA-4, VLA-4, VCAM-1, LFA-1, ICAM-1, CD44,osteopontin (SPP1), or any combination thereof. In some embodiments,such multi-target gene silencing can be used to treat more than onedisease or condition, if so needed.

In some embodiments, the sd-rxRNA targets MAP4K4. MAP4K4 is a mammalianserine/threonine protein kinase that belongs to a group of proteinkinases related to Saccharomyces cerevisiae Sterile 20 (STE20). MAP4K4(also known as NIK for Nck interacting kinase) was first identified in amouse screen for proteins that interact with the SH3 domain of Nck (Suet al. (1997). Since its discovery, MAP4K4 has been and continues to belinked to wide range of physiological functions.

Approaches for RNAi-mediated inhibition of MAP4K4 expression aredescribed in, and incorporated by reference from, U.S. ProvisionalApplication Ser. No. 61/199,661, entitled “Inhibition of MAP4K4 throughRNAi,” filed on Nov. 19, 2008, and PCT application PCT/US2009/006211,filed on Nov. 19, 2009 and entitled “Inhibition of MAP4K4 through RNAi.”sd-rxRNA molecules targeting MAP4K4 are compatible with aspects of theinvention. In some embodiments an sd-rxRNA molecule targeting VEGF andan sd-rxRNA molecule targeting MAP4K4 can be administered together.

Table 1 presents non-limiting examples of sd-rxRNA targets and areas inwhich they can be applied.

TABLE 1 Examples of sd-rxRNA targets and applications Target Area ofInterest Possible Indications VEGF Neovascularization i) AMD/DME Map4K4Inflammation i) Geographic Atrophy CTGF Angiogenesis,/ i) AMD/DMEFibrosis ii) Proliferative Vitreoretinopathy Scarring iii) Prevention ofTrabeculectomy Failure PTGS2 Inflammation i) Cystoid Macular Edema (Post(COX-2) Surgery), ii) Geographic Atrophy TGFβ Fibrosis/Scarring i)Proliferative Vitreoretinopathy ii) Prevention of Trabeculectomy Failureiii) Diabetic Retinopathy VEGF/COX-2 Neovascularization/ i) AMD/DMEinflamation ii) Geographic Atrophy iii) Proliferative Vitreoretinopathyiv) Prevention of Trabeculectomy Failure VEGF/CTGF Neovascularization/i) AMD/DME fibrosis ii) Geographic Atrophy iii) ProliferativeVitreoretinopathy iv) Prevention of Trabeculectomy Failure VEGF/Neovascularization/ i) AMD/DME MAP4K4 inflamation ii) Geographic Atrophyiii) Proliferative Vitreoretinopathy iv) Prevention of TrabeculectomyFailure

In one embodiment, in vitro treatment of cells with oligonucleotides canbe used for ex vivo therapy of cells removed from a subject or fortreatment of cells which did not originate in the subject, but are to beadministered to the subject (e.g., to eliminate transplantation antigenexpression on cells to be transplanted into a subject). In addition, invitro treatment of cells can be used in non-therapeutic settings, e.g.,to evaluate gene function, to study gene regulation and proteinsynthesis or to evaluate improvements made to oligonucleotides designedto modulate gene expression or protein synthesis. In vivo treatment ofcells can be useful in certain clinical settings where it is desirableto inhibit the expression of a protein. The subject nucleic acids can beused in RNAi-based therapy in any animal having RNAi pathway, such ashuman, non-human primate, non-human mammal, non-human vertebrates,rodents (mice, rats, hamsters, rabbits, etc.), domestic livestockanimals, pets (cats, dogs, etc.), Xenopus, fish, insects (Drosophila,etc.), and worms (C. elegans), etc.

The invention provides methods for inhibiting or preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject anucleic acid of the invention. If appropriate, subjects are firsttreated with a priming agent so as to be more responsive to thesubsequent RNAi therapy. Subjects at risk for a disease which is causedor contributed to by aberrant or unwanted target gene expression oractivity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays known in the art. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the target gene aberrancy, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.Depending on the type of target gene aberrancy, for example, a targetgene, target gene agonist or target gene antagonist agent can be usedfor treating the subject.

In another aspect, the invention pertains to methods of modulatingtarget gene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the methods of theinvention involve contacting a cell capable of expressing target genewith a nucleic acid of the invention that is specific for the targetgene or protein (e.g., is specific for the mRNA encoded by said gene orspecifying the amino acid sequence of said protein) such that expressionor one or more of the activities of target protein is modulated. Thesemethods can be performed in vitro (e.g., by culturing the cell with theagent), in vivo (e.g., by administering the agent to a subject), or exvivo. The subjects may be first treated with a priming agent so as to bemore responsive to the subsequent RNAi therapy if desired. As such, thepresent invention provides methods of treating a subject afflicted witha disease or disorder characterized by aberrant or unwanted expressionor activity of a target gene polypeptide or nucleic acid moleculeInhibition of target gene activity is desirable in situations in whichtarget gene is abnormally unregulated and/or in which decreased targetgene activity is likely to have a beneficial effect.

Thus the therapeutic agents of the invention can be administered tosubjects to treat (prophylactically or therapeutically) disordersassociated with aberrant or unwanted target gene activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent. Pharmacogenomics deals with clinically significanthereditary variations in the response to drugs due to altered drugdisposition and abnormal action in affected persons.

For the purposes of the invention, ranges may be expressed herein asfrom “about” one particular value, and/or to “about” another particularvalue. When such a range is expressed, another embodiment includes fromthe one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “aprotein” or “a nucleic acid molecule” refers to one or more of thosecompounds or at least one compound. As such, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably. Furthermore, a compound “selectedfrom the group consisting of” refers to one or more of the compounds inthe list that follows, including mixtures (i.e., combinations) of two ormore of the compounds. According to the present invention, an isolated,or biologically pure, protein or nucleic acid molecule is a compoundthat has been removed from its natural milieu. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which thecompound has been purified. An isolated compound of the presentinvention can be obtained from its natural source, can be produced usingmolecular biology techniques or can be produced by chemical synthesis.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1 Ocular Administration of sd-rxRNA Molecules

Subretinal and intravitreal administration was found to be highlyeffective in delivering sd-rxRNA molecules to all of the cell types inthe eye. sd-rxRNA targeting MAP4K4 or non-targeting (with or without aDY547 label) were injected by either subretinal or intravitrealadministration. 5 and 10 μg doses were evaluated. No signs ofinflammation were detected. Eyes looked healthy and no signs of cellularmigration (indicative of inflammatory response) were observed.

Compound uptake was visualized by fundus microscopy. Mice weresacrificed at 24 and 48 hours, retinas were collected, fixed, andparaffin embedded and compound uptake and distribution was analyzed byconfocal microscopy.

FIG. 1 demonstrates a confocal triple overlay of DIC, DY547 and Hoechst.As revealed by the staining, by 24 hours, sd-rxRNA had penetrated theentire retina. FIG. 2 provides a comparison of delivery of sd-rxRNA withtraditional RNAi compounds. 10 μg of RNA in 1 μl of PBS was deliveredintravitreally. White light fundus imaging verified that the retinas hadnormal ocular architecture with no hemorrhage or gross signs ofinflammation. Fluorescent images were taken with a fundus camera (acamera with a low power microscope designed to photograph the interiorsurface of the eye). As revealed in FIG. 2, both sd-rxRNA andtraditional RNAi compounds were detected in the retina immediately afteradministration. However, after 24 and 48 hours, only the sd-rxRNAmolecule was detected.

FIG. 3 reveals that sd-rxRNA was detected throughout the retinafollowing intravitreal dosing of sd-rxRNA, but not following dosing ofPBS or traditional RNAi compounds.

FIG. 4 reveals that sd-rxRNA penetrated the retina to the outer segmentsof the photoreceptors.

FIG. 5 reveals that in ARPE-19 cells, sd-rxRNA shows robust uptake andsilencing compared to rxRNAori.

FIG. 6 shows some non-limiting examples of sd-rxRNAs for use in ocularindiciations.

The effectiveness of sd-rxRNA directed to VEGF is demonstrated in FIG. 7which shows the percentage of VEGF mRNA levels (normalized to PPIB)after doses of the VEGF-specific sd-rxRNA.

FIG. 8 reveals efficient penetration of sd-rxRNA following intravitrealdosing. Fluorescently-labeled RNAi compounds were administered to mouseeye using 1 μl intravitreal injection. By 2-3 hrs post injection,sd-rxRNA was present in the ganglion cell layer and was present in theouter segments of the photoreceptors by 24 hrs post dosing. Fluorescencewas detected using laser scanning confocal microscopy.

FIG. 9 reveals significant retinal uptake of sd-rxRNA but not rxRNAoriin vivo at 24 h. Fluorescently-tagged RNAi compounds were administeredand whole eye mounts were prepared from eyes harvested at 24 h postdose. Whole eyes were fixed in 4% paraformaldehyde overnight followed byformalin fixing and paraffin embedding. Transverse sections were cut (5μm thick), stained with Hoechst and visualized using a Leica SP5Confocal Microscope. Images shown are a triple overlay of the Hoechst,DY547 (red tag on RNAi compound), and differential interference contract(DIC) images. Panel B shows a higher magnification view (scale bar is 10μm) from the sd-rxRNA sample shown in panel A, revealing delivery to theretinal pigment epithelium cells.

FIGS. 10 and 11 reveal significant retinal uptake of sd-rxRNA in rabbitretina 24 h post dose. Fluorescently-labeled RNAi compounds (100 ug)were administered to rabbit eye via single intravitreal injection. At 24h post injection whole eyes were harvested and frozen in optimal cuttingtemperature (OCT) gel. Frozen blocks were cut in sections were stainedwith Hoechst. sd-rxRNA was present throughout the retinal cell layersfollowing intravitreal dosing. Fluorescence was detected using laserscanning confocal microscopy.

FIG. 12 reveals significant silencing of target mRNA following singleintravitreal injection. sd-rxRNA was administered by intravitrealinjection (in 1 ul) to mouse eyes at indicated doses. Retinas wereharvested at 48 hours, and mRNA levels quantified by QPCR and normalizedto b-actin, n=5-8 (data from different studies was included, graphed+/−SD relative to NTC, * p≦0.05, ** p≦0.01).

FIG. 13 reveals significant silencing of target mRNA following singleintravitreal injection. 3 ug of PPIB or NTC sd-rxRNAs were administeredto mouse eyes by single intravitreal injection (1 ul). Retinas wereharvested at 1, 2, 4, 7, 14, 21 and 28 days post injection. mRNA levelswere quantified by qPCR and normalized to b-actin. Data was assembledfrom 6 different studies to enable sufficient ‘n’ for each data point(n=5-8) (graphed +/−SD relative to PBS in each study, * p≦0.01)

FIG. 14 reveals significant silencing of target mRNA following singleintravitreal injection. 3 ug of Map4K4 or NTC sd-rxRNAs wereadministered to mouse eyes by single intravitreal injection (1 ul).Retinas were harvested at 1, 2, 4, 7, 14, 21 and 28 days post injection.mRNA levels were quantified by qPCR and normalized to b-actin. Data wasassembled from 6 different studies to enable sufficient ‘n’ for eachdata point (n=5-8) (graphed +/−SD relative to PBS in each study, *p≦0.05, * * p≦0.01).

FIG. 15 reveals that sd-rxRNA did not induce blood vessel leakage threeweeks post dose. sd-rxRNA was administered by single intravitreal dosing(1 ul) of 5 μg of both Map4k4-TEG and Map4k4-HP sd-rxRNA and PBS.Fluorescein-labeled dextran was administered subcutaneously prior tofluorescent fundus imaging 1, 2, and 3 weeks post dosing. Fluoresceinangiography revealed no leakage of retinal blood vessels.

FIG. 16 reveals that sd-rxRNA did not induce retinal architecturaldamage three weeks post dose. sd-rxRNA was administered by singleintravitreal dosing (1 ul) of 5 μg of both Map4k4-TEG and Map4k4-HPsd-rxRNA and PBS. Optical coherence tomography was performed 2, and 3weeks post dosing. Representative images show normal retinalarchitecture and thickness.

FIG. 17 reveals that sd-rxRNA did not impair retinal function threeweeks post dose. sd-rxRNA was administered by single intravitreal dosing(1 ul) of 5 μg of both Map4k4-TEG and Map4k4-HP sd-rxRNA and PBS.Scotopic electroretinography (ERG) recordings were taken three weekspost dosing and revealed similar retinal function following dosing withsd-rxRNA and PBS. Photopic recordings were also collected with similaroutcome.

FIG. 18 reveals a multi-targeted silencing approach with sd-rxRNAs. Thesilencing activity of several sd-rxRNAs was determined in cells treatedwith up to 4 sd-rxRNAs targeting multiple genes of interest. When dosedin combination, sd-rxRNAs retained their potent efficacy, demonstratingtheir potential for multi-targeting silencing.

Table 2 presents 25-mer sequences within VEGF. rxRNAoris and sd-rxRNAscan be directed against sequences within Table 2. In some embodiments,rxRNAoris comprise the sequences presented in Table 2.

Table 3 presents non-limiting examples of sd-rxRNA molecules directedagainst SPP1.

Table 4 presents non-limiting examples of sd-rxRNA sequences directedagainst PTGS2 (COX-2).

Table 5 presents non-limiting examples of sd-rxRNA sequences directedagainst CTGF.

Table 6 presents non-limiting examples of sd-rxRNA sequences directedagainst TGFβ2.

Table 7 presents non-limiting examples of sd-rxRNA sequences directedagainst TGFβ1.

Example 2 Identification of sd-rxRNAs Targeting VEGF

Optimal sequences for sd-rxRNA development were identified using asequence selection algorithm (Table 2). The algorithm selects sequencesbased on the following criteria: a GC content greater than 32% but lessthan 47%, homology to specific animal models (e.g., mouse or rat),avoidance of 5 or more U/U stretches and/or 2 or more G/C stretches, anoff-target hit score of less than 500, and avoidance of sequencescontained within the 5′ UTR.

The sequences were developed initially as 25 nucleotide blunt-endedduplexes with O-methyl modification. Such sequences were screened invarious cell lines to identify those were most efficient in reducinggene expression. Several concentrations of the RNA molecules, such as0.025, 0.1 and 0.25 nM, were tested, and suitable concentrations weredetermined Dose response curves were generated to determine the mostpotent sequences. Those sequences were developed into sd-rxRNA moleculesbased on parameters described throughout the application.

Table 8 presents non-limiting examples of sd-rxRNA sequences directedagainst VEGF.

Table 9 presents non-limiting examples of rxRNAori sequences directedagainst VEGF.

Table 10 presents results of optimization of sd-rxRNA sequences directedagainst VEGF, using a variety of chemical modification patterns.

Example 3 Linker Chemistry

FIG. 19 demonstrates that variation of linker chemistry does notinfluence silencing activity of sd-rxRNAs in vitro. Two different linkerchemistries were evaluated, a hydroxyproline linker and ribo linker, onmultiple sd-rxRNAs (targeting Map4k4 or PPIB) in passive uptake assaysto determine linkers which favor self delivery. HeLa cells weretransfected in the absence of a delivery vehicle (passive transfection)with sd-rxRNAs at 1 uM, 0.1 uM or 0.01 uM for 48 hrs. Use of eitherlinker results in an efficacious delivery of sd-rxRNA.

The ribo linker used in Example 1 had the following structure:

TABLE 2  hVEGF stealth sequences % Remaining Expression as Oligo GeneRef SEQ 25-mer Sense Strand Compared to ID Region Pos ID NO(position 25 of SS, replaced with A) NTC (100 pM) 18832 3′UTR 3471   1UAUCAUUUAUUUAUUGGUGCUACUA  37% 18811 3′UTR 3199   2UUAAUUUUGCUAACACUCAGCUCUA  37% 18902 3′UTR 2792   3CCUCACACCAUUGAAACCACUAGUA  39% 18830 3′UTR 3429   4CUACAUACUAAAUCUCUCUCCUUUA  41% 18880 CDS 1343   5CCAACAUCACCAUGCAGAUUAUGCA  42% 18756 CDS 1389   6GCACAUAGGAGAGAUGAGCUUCCUA  42% 18913 3′UTR 3163   7AUCGGUGACAGUCACUAGCUUAUCA  42% 18909 3′UTR 3073   8UUUAUGAGAUGUAUCUUUUGCUCUA  42% 18831 3′UTR 3430   9UACAUACUAAAUCUCUCUCCUUUUA  43% 18778 3′UTR 2183  10UAACAGUGCUAAUGUUAUUGGUGUA  43% 18793 3′UTR 2932  11UUGUGGAGGCAGAGAAAAGAGAAAA  43% 18898 3′UTR 2210  12CACUGGAUGUAUUUGACUGCUGUGA  46% 18760 3′UTR 1853  13AUCACCAUCGACAGAACAGUCCUUA  46% 18766 3′UTR 1859  14AUCGACAGAACAGUCCUUAAUCCAA  46% 18908 3′UTR 3072  15AUUUAUGAGAUGUAUCUUUUGCUCA  46% 18903 3′UTR 2794  16UCACACCAUUGAAACCACUAGUUCA  46% 18834 3′UTR 3476  17UUUAUUUAUUGGUGCUACUGUUUAA  47% 18828 3′UTR 3427  18UUCUACAUACUAAAUCUCUCUCCUA  48% 18761 3′UTR 1854  19UCACCAUCGACAGAACAGUCCUUAA  48% 18892 3′UTR 1985  20CCUCUUGGAAUUGGAUUCGCCAUUA  48% 18764 3′UTR 1857  21CCAUCGACAGAACAGUCCUUAAUCA  48% 18883 CDS 1347  22CAUCACCAUGCAGAUUAUGCGGAUA  48% 18790 3′UTR 2790  23GUCCUCACACCAUUGAAACCACUAA  48% 18912 3′UTR 3162  24GAUCGGUGACAGUCACUAGCUUAUA  49% 18794 3′UTR 2933  25UGUGGAGGCAGAGAAAAGAGAAAGA  49% 18900 3′UTR 2447  26AGGUCAGACGGACAGAAAGACAGAA  49% 18792 3′UTR 2931  27AUUGUGGAGGCAGAGAAAAGAGAAA  49% 18886 CDS 1352  28CCAUGCAGAUUAUGCGGAUCAAACA  49% 18769 3′UTR 1863  29ACAGAACAGUCCUUAAUCCAGAAAA  50% 18817 3′UTR 3252  30CACAUUCCUUUGAAAUAAGGUUUCA  50% 18865 3′UTR 1852  31CAUCACCAUCGACAGAACAGUCCUA  50% 18879 CDS 1342  32UCCAACAUCACCAUGCAGAUUAUGA  50% 18866 3′UTR 2926  33UGCCCAUUGUGGAGGCAGAGAAAAA  50% 18751 CDS 1356  34GCAGAUUAUGCGGAUCAAACCUCAA  50% 18899 3′UTR 2211  35ACUGGAUGUAUUUGACUGCUGUGGA  51% 18762 3′UTR 1855  36CACCAUCGACAGAACAGUCCUUAAA  51% 18777 3′UTR 2182  37UUAACAGUGCUAAUGUUAUUGGUGA  51% 18887 CDS 1353  38CAUGCAGAUUAUGCGGAUCAAACCA  51% 18846 3′UTR 3516  39GGAAAAGAUAUUAACAUCACGUCUA  52% 18877 CDS 1340  40AGUCCAACAUCACCAUGCAGAUUAA  52% 18813 3′UTR 3246  41CCAGCACACAUUCCUUUGAAAUAAA  53% 18810 3′UTR 3197  42AUUUAAUUUUGCUAACACUCAGCUA  53% 18798 3′UTR 2949  43AGAGAAAGUGUUUUAUAUACGGUAA  54% 18759 CDS 1396  44GGAGAGAUGAGCUUCCUACAGCACA  54% 18795 3′UTR 2935  45UGGAGGCAGAGAAAAGAGAAAGUGA  54% 18819 3′UTR 3363  46UGAUAAAAUAGACAUUGCUAUUCUA  54% 18916 3′UTR 3167  47GUGACAGUCACUAGCUUAUCUUGAA  55% 18836 3′UTR 3478  48UAUUUAUUGGUGCUACUGUUUAUCA  55% 18785 3′UTR 2191  49CUAAUGUUAUUGGUGUCUUCACUGA  56% 18874 CDS 1337  50AGGAGUCCAACAUCACCAUGCAGAA  56% 18750 CDS 1354  51AUGCAGAUUAUGCGGAUCAAACCUA  57% 18878 CDS 1341  52GUCCAACAUCACCAUGCAGAUUAUA  57% 18791 3′UTR 2930  53CAUUGUGGAGGCAGAGAAAAGAGAA  58% 18770 3′UTR 1884  54AAACCUGAAAUGAAGGAAGAGGAGA  58% 18776 3′UTR 2181  55AUUAACAGUGCUAAUGUUAUUGGUA  58% 18780 3′UTR 2185  56ACAGUGCUAAUGUUAUUGGUGUCUA  59% 18805 3′UTR 3155  57UCUCCCUGAUCGGUGACAGUCACUA  59% 18829 3′UTR 3428  58UCUACAUACUAAAUCUCUCUCCUUA  59% 18767 3′UTR 1860  59UCGACAGAACAGUCCUUAAUCCAGA  60% 18809 3′UTR 3196  60UAUUUAAUUUUGCUAACACUCAGCA  60% 18816 3′UTR 3251  61ACACAUUCCUUUGAAAUAAGGUUUA  60% 18867 CDS 1214  62CCCUGGUGGACAUCUUCCAGGAGUA  60% 18774 3′UTR 1987  63UCUUGGAAUUGGAUUCGCCAUUUUA  61% 18882 CDS 1346  64ACAUCACCAUGCAGAUUAUGCGGAA  61% 18905 3′UTR 2797  65CACCAUUGAAACCACUAGUUCUGUA  61% 18754 CDS 1385  66GCCAGCACAUAGGAGAGAUGAGCUA  61% 18822 3′UTR 3366  67UAAAAUAGACAUUGCUAUUCUGUUA  62% 18763 3′UTR 1856  68ACCAUCGACAGAACAGUCCUUAAUA  62% 18863 3′UTR 3589  69UAAACAACGACAAAGAAAUACAGAA  62% 18835 3′UTR 3477  70UUAUUUAUUGGUGCUACUGUUUAUA  63% 18893 3′UTR 2009  71UUAUUUUUCUUGCUGCUAAAUCACA  63% 18771 3′UTR 1885  72AACCUGAAAUGAAGGAAGAGGAGAA  63% 18894 3′UTR 2010  73UAUUUUUCUUGCUGCUAAAUCACCA  64% 18765 3′UTR 1858  74CAUCGACAGAACAGUCCUUAAUCCA  64% 18796 3′UTR 2936  75GGAGGCAGAGAAAAGAGAAAGUGUA  65% 18797 3′UTR 2946  76AAAAGAGAAAGUGUUUUAUAUACGA  65% 18821 3′UTR 3365  77AUAAAAUAGACAUUGCUAUUCUGUA  65% 18823 3′UTR 3367  78AAAAUAGACAUUGCUAUUCUGUUUA  66% 18869 CDS 1231  79CAGGAGUACCCUGAUGAGAUCGAGA  67% 18781 3′UTR 2187  80AGUGCUAAUGUUAUUGGUGUCUUCA  67% 18775 3′UTR 2180  81AAUUAACAGUGCUAAUGUUAUUGGA  67% 18870 CDS 1232  82AGGAGUACCCUGAUGAGAUCGAGUA  68% 18815 3′UTR 3248  83AGCACACAUUCCUUUGAAAUAAGGA  68% 18804 3′UTR 3135  84AUUCAUGUUUCCAAUCUCUCUCUCA  69% 18799 3′UTR 2950  85GAGAAAGUGUUUUAUAUACGGUACA  69% 18779 3′UTR 2184  86AACAGUGCUAAUGUUAUUGGUGUCA  69% 18924 3′UTR 3545  87UCUAGUGCAGUUUUUCGAGAUAUUA  69% 18758 CDS 1394  88UAGGAGAGAUGAGCUUCCUACAGCA  70% 18782 3′UTR 2188  89GUGCUAAUGUUAUUGGUGUCUUCAA  70% 18833 3′UTR 3475  90AUUUAUUUAUUGGUGCUACUGUUUA  70% 18800 3′UTR 3094  91UCUCUCUUGCUCUCUUAUUUGUACA  70% 18904 3′UTR 2795  92CACACCAUUGAAACCACUAGUUCUA  70% 18845 3′UTR 3515  93GGGAAAAGAUAUUAACAUCACGUCA  71% 18884 CDS 1348  94AUCACCAUGCAGAUUAUGCGGAUCA  71% 18818 3′UTR 3356  95GUGAUUCUGAUAAAAUAGACAUUGA  71% 18814 3′UTR 3247  96CAGCACACAUUCCUUUGAAAUAAGA  71% 18801 3′UTR 3131  97UAAAAUUCAUGUUUCCAAUCUCUCA  71% 18873 CDS 1236  98GUACCCUGAUGAGAUCGAGUACAUA  72% 18802 3′UTR 3133  99AAAUUCAUGUUUCCAAUCUCUCUCA  72% 18787 3′UTR 2212 100CUGGAUGUAUUUGACUGCUGUGGAA  72% 18854 3′UTR 3525 101AUUAACAUCACGUCUUUGUCUCUAA  72% 18901 3′UTR 2791 102UCCUCACACCAUUGAAACCACUAGA  73% 18753 CDS 1384 103GGCCAGCACAUAGGAGAGAUGAGCA  73% 18820 3′UTR 3364 104GAUAAAAUAGACAUUGCUAUUCUGA  73% 18807 3′UTR 3194 105GAUAUUUAAUUUUGCUAACACUCAA  73% 18772 3′UTR 1886 106ACCUGAAAUGAAGGAAGAGGAGACA  74% 18803 3′UTR 3134 107AAUUCAUGUUUCCAAUCUCUCUCUA  74% 18844 3′UTR 3514 108GGGGAAAAGAUAUUAACAUCACGUA  75% 18888 CDS 1411 109CUACAGCACAACAAAUGUGAAUGCA  75% 18895 3′UTR 2077 110ACACACCCACCCACAUACAUACAUA  76% 18858 3′UTR 3553 111AGUUUUUCGAGAUAUUCCGUAGUAA  77% 18889 3′UTR 1981 112GGUCCCUCUUGGAAUUGGAUUCGCA  77% 18856 3′UTR 3551 113GCAGUUUUUCGAGAUAUUCCGUAGA  78% 18931 3′UTR 3588 114UUAAACAACGACAAAGAAAUACAGA  78% 18808 3′UTR 3195 115AUAUUUAAUUUUGCUAACACUCAGA  78% 18825 3′UTR 3423 116AGAAUUCUACAUACUAAAUCUCUCA  78% 18864 3′UTR 3590 117AAACAACGACAAAGAAAUACAGAUA  78% 18881 CDS 1345 118AACAUCACCAUGCAGAUUAUGCGGA  79% 18906 3′UTR 2798 119ACCAUUGAAACCACUAGUUCUGUCA  79% 18868 CDS 1229 120UCCAGGAGUACCCUGAUGAGAUCGA  79% 18897 3′UTR 2196 121GUUAUUGGUGUCUUCACUGGAUGUA  79% 18788 3′UTR 2213 122UGGAUGUAUUUGACUGCUGUGGACA  79% 18896 3′UTR 2195 123UGUUAUUGGUGUCUUCACUGGAUGA  79% 18784 3′UTR 2190 124GCUAAUGUUAUUGGUGUCUUCACUA  79% 18847 3′UTR 3518 125AAAAGAUAUUAACAUCACGUCUUUA  80% 18852 3′UTR 3523 126AUAUUAACAUCACGUCUUUGUCUCA  80% 18850 3′UTR 3521 127AGAUAUUAACAUCACGUCUUUGUCA  80% 18917 3′UTR 3264 128AAAUAAGGUUUCAAUAUACAUCUAA  81% 18871 CDS 1234 129GAGUACCCUGAUGAGAUCGAGUACA  81% 18837 3′UTR 3479 130AUUUAUUGGUGCUACUGUUUAUCCA  81% 18910 3′UTR 3130 131AUAAAAUUCAUGUUUCCAAUCUCUA  81% 18875 CDS 1338 132GGAGUCCAACAUCACCAUGCAGAUA  82% 18923 3′UTR 3544 133CUCUAGUGCAGUUUUUCGAGAUAUA  82% 18853 3′UTR 3524 134UAUUAACAUCACGUCUUUGUCUCUA  82% 18876 CDS 1339 135GAGUCCAACAUCACCAUGCAGAUUA  82% 18824 3′UTR 3422 136GAGAAUUCUACAUACUAAAUCUCUA  84% 18768 3′UTR 1862 137GACAGAACAGUCCUUAAUCCAGAAA  84% 18891 3′UTR 1983 138UCCCUCUUGGAAUUGGAUUCGCCAA  85% 18842 3′UTR 3484 139UUGGUGCUACUGUUUAUCCGUAAUA  85% 18838 3′UTR 3480 140UUUAUUGGUGCUACUGUUUAUCCGA  86% 18925 3′UTR 3546 141CUAGUGCAGUUUUUCGAGAUAUUCA  87% 18859 3′UTR 3554 142GUUUUUCGAGAUAUUCCGUAGUACA  88% 18885 CDS 1351 143ACCAUGCAGAUUAUGCGGAUCAAAA  88% 18857 3′UTR 3552 144CAGUUUUUCGAGAUAUUCCGUAGUA  88% 18849 3′UTR 3520 145AAGAUAUUAACAUCACGUCUUUGUA  88% 18755 CDS 1387 146CAGCACAUAGGAGAGAUGAGCUUCA  88% 18927 3′UTR 3548 147AGUGCAGUUUUUCGAGAUAUUCCGA  88% 18786 3′UTR 2194 148AUGUUAUUGGUGUCUUCACUGGAUA  89% 18926 3′UTR 3547 149UAGUGCAGUUUUUCGAGAUAUUCCA  91% 18928 3′UTR 3549 150GUGCAGUUUUUCGAGAUAUUCCGUA  91% 18757 CDS 1391 151ACAUAGGAGAGAUGAGCUUCCUACA  91% 18848 3′UTR 3519 152AAAGAUAUUAACAUCACGUCUUUGA  92% 18921 3′UTR 3542 153GUCUCUAGUGCAGUUUUUCGAGAUA  93% 18907 3′UTR 3070 154CUAUUUAUGAGAUGUAUCUUUUGCA  93% 18783 3′UTR 2189 155UGCUAAUGUUAUUGGUGUCUUCACA  93% 18918 3′UTR 3296 156AUAUAUAUUUGGCAACUUGUAUUUA  93% 18851 3′UTR 3522 157GAUAUUAACAUCACGUCUUUGUCUA  94% 18890 3′UTR 1982 158GUCCCUCUUGGAAUUGGAUUCGCCA  95% 18827 3′UTR 3425 159AAUUCUACAUACUAAAUCUCUCUCA  95% 18812 3′UTR 3241 160GCUCCCCAGCACACAUUCCUUUGAA  96% 18773 3′UTR 1887 161CCUGAAAUGAAGGAAGAGGAGACUA  97% 18855 3′UTR 3526 162UUAACAUCACGUCUUUGUCUCUAGA  98% 18789 3′UTR 2214 163GGAUGUAUUUGACUGCUGUGGACUA  98% 18826 3′UTR 3424 164GAAUUCUACAUACUAAAUCUCUCUA  98% 18919 3′UTR 3297 165UAUAUAUUUGGCAACUUGUAUUUGA  98% 18752 CDS 1381 166CAAGGCCAGCACAUAGGAGAGAUGA  99% 18914 3′UTR 3165 167CGGUGACAGUCACUAGCUUAUCUUA 100% 18930 3′UTR 3587 168UUUAAACAACGACAAAGAAAUACAA 101% 18911 3′UTR 3161 169UGAUCGGUGACAGUCACUAGCUUAA 103% 18872 CDS 1235 170AGUACCCUGAUGAGAUCGAGUACAA 105% 18929 3′UTR 3550 171UGCAGUUUUUCGAGAUAUUCCGUAA 106% 18860 3′UTR 3555 172UUUUUCGAGAUAUUCCGUAGUACAA 107% 18839 3′UTR 3481 173UUAUUGGUGCUACUGUUUAUCCGUA 109% 18806 3′UTR 3160 174CUGAUCGGUGACAGUCACUAGCUUA 112% 18843 3′UTR 3491 175UACUGUUUAUCCGUAAUAAUUGUGA 114% 18861 3′UTR 3556 176UUUUCGAGAUAUUCCGUAGUACAUA 118% 18841 3′UTR 3483 177AUUGGUGCUACUGUUUAUCCGUAAA 118% 18922 3′UTR 3543 178UCUCUAGUGCAGUUUUUCGAGAUAA 120% 18915 3′UTR 3166 179GGUGACAGUCACUAGCUUAUCUUGA 122% 18920 3′UTR 3298 180AUAUAUUUGGCAACUUGUAUUUGUA 125% 18840 3′UTR 3482 181UAUUGGUGCUACUGUUUAUCCGUAA 127% 18862 3′UTR 3557 182UUUCGAGAUAUUCCGUAGUACAUAA 135%

TABLE 3  SPP1 (Accession Number NM_000582.2) sd-rxRNA sequences% remaining Oligo Start SEQ ID SEQ expression Number Site NOSense sequence ID NO Antisense sequence (1 uM A549) 14084 1025 183mC.mU.mC. A.mU. G. A. 184 P.mU.fC.fU. A. A.fU.fU.fC.  61%A.mU.mU. A. G. A.Chl A.fU. G. A. G* A* A* A*mU* A* C. 14085 1049 185mC.mU. G. A. G. 186 P.mU. A. A.fU.fU. G.  50% G.mU.mC. A. A.mU.mU.A.fC.fC.fU.mC. A. G* A* A.Chl A* G* A*mU* G. 14086 1051 187G. A. G. G.mU.mC. A. 188 P.mU.fU.fU. A. A.fU.fU. G. n/aA.mU.mU. A. A. A.Chl A.fC.mC.mU.mC* A* G* A* A* G* A. 14087 1048 189mU.mC.mU. G. A. G. 190 P.mA. A.fU.fU. G.  69% G.mU.mC. A.A.fC.fC.fU.fC. A. G. A* A* A.mU.mU.Chl G* A*mU* G* C.   14088 1050 191mU. G. A. G. G.mU.mC. 192 P.mU.fU. A. A.fU.fU. G.  76%A. A.mU.mU. A. A.Chl A.fC.fC.mU.mC. A* G* A* A* G* A* U. 14089 1047 193mU.mU.mC.mU. G. A. 194 P.mA.fU.fU. G.  60% G. G.mU.mC. A.A.fC.fC.fU.fC. A. G. A. A* A.mU.Chl G* A*mU* G*mC* A. 14090 800 195G.mU.mC. A. G.mC.mU. 196 P.mU.fC. A.fU.fC.fC. A.  71%G. G. A.mU. G. A.Chl G.fC.fU. G. A.mC*mU*mC* G*mU*mU* U.   14091 492 197mU.mU.mC.mU. G. 198 P.mA. G. A.fU.fU.fC. n/a A.mU. G. A.A.fU.fC. A. G. A. A*mU* A.mU.mC.mU.Chl G* G*mU* G* A. 14092 612 199mU. G. G. A.mC.mU. G. 200 P.mU. G. A.fC.fC.fU.fC. A. n/aA. G. G.mU.mC. A.Chl G.fU.mC.mC. A*mU* A* A* A*mC* C. 14093 481 201G. A. G.mU.mC.mU.mC.  202 P.mA. A.fU. G. G.fU. G. A. n/aA.mC.mC. A.mU.mU.Chl G. A.mC.mU.mC* A*mU*mC* A* G* A. 14094 614 203G. A.mC.mU. G. A. G. 204 P.mU.fU.fU. G. n/a G.mU.mC. A. A. A.ChlA.fC.fC.fU.fC. A. G.mU.mC*mC* A*mU* A* A* A. 14095 951 205mU.mC. A.mC. A. 206 P.mU.fU.fC. A.fU. G.  89% G.mC.mC. A.mU. G. A.G.fC.fU. G.mU. G. A* A* A.Chl A*mU*mU*mC* A. 14096 482 207A. G.mU.mC.mU.mC. 208 P.mG. A. A.fU. G. G.fU. G.  87% A.mC.mC.A. G. A.mC.mU*mC* A.mU.mU.mC.Chl A*mU*mC* A* G. 14097 856 209A. A. G.mC. G. G. A. A. 210 P.mU. G.  88% A. G.mC.mC. A.ChlG.fC.fU.fU.fU.fC.fC. G.mC.mU.mU* A*mU* A*mU* A* A. 14098 857 211A. G.mC. G. G. A. A. A. 212 P.mU.fU. G. 113% G.mC.mC. A. A.ChlG.fC.fU.fU.fU.fC.fC. G.mC.mU*mU* A*mU* A*mU* A. 14099 365 213A.mC.mC. A.mC. A.mU. 214 P.mU.fC. A.fU.fC.fC. A.fU.  98%G. G. A.mU. G. A.Chl G.fU. G. G.mU*mC* A*mU* G* G* C. 14100 359 215G.mC.mC. A.mU. G. 216 P.mA.fU. G.fU. G. G.fU.fC.  84% A.mC.mC. A.mC.A.fU. G. A.mU.Chl G.mC*mU*mU*mU*mC* G* U. 14101 357 217A. A. G.mC.mC. A.mU. 218 P.mG.fU. G. G.fU.fC. A.fU.  88%G. A.mC.mC. A.mC.Chl G. G.mC.mU.mU*mU*mC* G*mU*mU* G. 14102 858 219G.mC. G. G. A. A. A. 220 P.mA.fU.fU. G. n/a G.mC.mC. A. A.mU.ChlG.fC.fU.fU.fU.fC.mC. G.mC*mU*mU* A*mU* A* U. 14103 1012 221A. A. A.mU.mU.mU.mC. 222 P.mA. A. A.fU. A.fC. G. A.  93% G.mU.A. A.mU.mU.mU*mC* A* A.mU.mU.mU.Chl G* G*mU* G. 14104 1014 223A.mU.mU.mU.mC. 224 P.mA. G. A. A. A.fU. A.fC.  89% G.mU. G. A. A.A.mU.mU.mU.mC.mU. A.mU*mU*mU*mC* A* Chl G* G. 14105 356 225A. A. A. G.mC.mC. 226 P.mU. G. G.fU.fC. A.fU. G.  85% A.mU. G. A.mC.mC.G.fC.mU.mU.mU*mC* A.Chl G*mU*mU* G* G. 14106 368 227 A.mC. A.mU. G. G.228 P.mA.fU. A.fU.fC.  67% A.mU. G. A.mU. A.fU.fC.fC. A.mU. G.mU*A.mU.Chl G* G*mU*mC* A* U. 14107 1011 229 G. A. A. 230P.mA. A.fU. A.fC. G. A. A.  87% A.mU.mU.mU.mC. A.fU.mU.mU.mC* A* G*G.mU. A.mU.mU.Chl G*mU* G* U. 14108 754 231 G.mC. 232P.mA. A.fU.fC. A. G. A. A.  73% G.mC.mC.mU.mU.mC.m G. G.mC. G.mC*U. G. A.mU.mU.Chl G*mU*mU*mC* A* G. 14109 1021 233 A.mU.mU.mU.mC.mU. 234P.mA.fU.fU.fC. A.fU. G. A. 128% mC. A.mU. G. A. G. A. A. A.mU* A*mC* G*A.mU.Chl A* A* A. 14110 1330 235 mC.mU.mC.mU.mC. 236P.mC.fU. A.fU.fU.fC. A.fU. 101% A.mU. G. A. A.mU. A.G. A. G. A. G* A* A*mU* G.Chl A* A* C. 14111 346 237A. A. G.mU.mC.mC. A. 238 P.mU.fU.fU.fC. G.fU.fU.  59%A.mC. G. A. A. A.Chl G. G. A.mC.mU.mU* A*mC*mU*mU* G* G. 14112 869 239A.mU. G. A.mU. G. A. G. 240 P.mU.fU. G.fC.fU.fC.fU.fC.  89%A. G.mC. A. A.Chl A.fU.mC. A.mU*mU* G* G*mC*mU* U. 14113 701 241G.mC. G. A. G. G. A. 242 P.mU.fU.fC. A.  95% G.mU.mU. G. A. A.ChlA.fC.fU.fC.fC.fU.mC. G.mC*mU*mU*mU*mC* mC* A. 14114 896 243mU. G. A.mU.mU. G. 244 P.mU. G. A.fC.fU. A.fU.fC.  87% A.mU. A. G.mU.mC.A. A.mU.mC. A*mC* A.Chl A*mU*mC* G* G. 14115 1035 245A. G. A.mU. A. G.mU. 246 P.mA. G. A.fU. G.fC.  82% G.mC. A.mU.mC.mU.ChlA.fC.fU. A.mU.mC.mU* A* A*mU*mU*mC* A. 14116 1170 247 A.mU. G.mU. G.mU.248 P.mA. A.fU. A. G. A.fU.  36% A.mU.mC.mU. A.fC. A.mC. A.mU.mU.ChlA.mU*mU*mC* A* A*mC* C. 14117 1282 249 mU.mU.mC.mU. A.mU. 250P. mU.fU.fC.fU.fU.fC.fU.  91% A. G. A. A. G. A. A.ChlA.fU. A. G. A. A*mU* G* A* A*mC* A. 14118 1537 251 mU.mU. G.mU.mC.mC.252 P.mA. A.fU.fU. G.fC.fU. G. 152% A. G.mC. A. G. A.mC. A. A*mC*mC*A.mU.mU.Chl G*mU* G* G. 14119 692 253 A.mC. A.mU. G. G. A. A. 254P.mU.fC. n/a A. G. C.mG. A.Chl G.fC.fU.fU.fU.fC.fC. A.mU.G.mU* G*mU* G* A* G* G. 14120 840 255 G.mC. A. G.mU.mC.mC. 256P.mU. A. A.fU.fC.fU. G. G.  87% A. G. A.mU.mU. A.ChlA.fC.mU. G.mC*mU*mU* G*mU* G* G. 14121 1163 257 mU. G. G.mU.mU. G. A.258 P.mA.fC. A.fC. A.fU.fU.fC.  31% A.mU. G.mU. G.mU.ChlA. A.mC.mC. A* A*mU* A* A* A* C. 14122 789 259 mU.mU. A.mU. G. A. A. 260P.mA.fC.fU.fC.  96% A.mC. G. A. G.mU.Chl G.fU.fU.fU.fC. A.mU. A.A*mC*mU* G*mU*mC* C. 14123 841 261 mC. A. G.mU.mC.mC. A. 262P.mA.fU. A. A.fU.fC.fU. G. 110% G. A.mU.mU. A.mU.Chl G. A.mC.mU.G*mC*mU*mU* G*mU* G. 14124 852 263 A.mU. A.mU. A. A. 264P.mU.fU.fU.fC.fC.  91% G.mC. G. G. A. A. A.Chl G.fC.fU.fU. A.mU. A.mU*A* A*mU*mC*mU* G. 14125 209 265 mU. A.mC.mC. A. 266 P.mU. G.fU.fU.fU. A.110% G.mU.mU. A. A. A.mC. A.fC.fU. G. G.mU. A*mU* A.Chl G* G*mC* A* C.14126 1276 267 mU. G.mU.mU.mC. 268 P.mU. A.fU. A. G. A. A.fU. n/aA.mU.mU.mC.mU. G. A. A.mC. A*mU* A* G* A.mU. A.Chl A*mC* A. 14127 137269 mC.mC. G. A.mC.mC. A. 270 P.mU.fU.fU.fC.fC.fU.fU.  71%A. G. G. A. A. A.Chl G. G.fU.mC. G. G*mC* G*mU*mU*mU* G. 14128 711 271G. A. A.mU. G. G.mU. 272 P.mG.fU. A.fU. G.fC. 115% G.mC. A.mU. A.mC.ChlA.fC.fC. A.mU.mU.mC* A* A*mC*mU*mC* C. 14129 582 273A.mU. A.mU. G. A.mU. 274 P.mU.fC. G. G.fC.fC.  97% G. G.mC.mC. G. A.ChlA.fU.fC. A.mU. A.mU* G*mU* G*mU*mC* U. 14130 839 275 A. G.mC. A. 276P.mA. A.fU.fC.fU. G. G. 102% G.mU.mC.mC. A. G. A.fC.fU. G.mC.mU*mU*A.mU.mU.Chl G*mU* G* G* C. 14131 1091 277 G.mC. A.mU.mU.mU. A. 278P.mU.fU.fU. G. A.fC.fU. A.  10% G.mU.mC. A. A. A.ChlA. A.mU. G.mC* A* A* A* G*mU* G. 14132 884 279 A. G.mC. 280P.mA.fC. A.fU.fC. G. G. A.  93% A.mU.mU.mC.mC. G. A.fU. G.mC.mU*mC*A.mU. G.mU.Chl A*mU*mU* G* C. 14133 903 281 mU. A. G.mU.mC. A. G. 282P.mA. A. G.fU.fU.fC.fC.fU.  97% G. A. A.mC.mU.mU.ChlG. A.mC.mU. A*mU*mC* A* A*mU* C. 14134 1090 283 mU. G.mC. 284P.mU.fU. G. A.fC.fU. A. A.  39% A.mU.mU.mU. A. A.fU. G.mC. A* A* A*G.mU.mC. A. A.Chl G*mU* G* A. 14135 474 285 G.mU.mC.mU. G. 286P.mA. G. A.fC.fU.fC.  99% A.mU. G. A. A.fU.fC. A. G. A.mC*mU*G.mU.mC.mU.Chl G* G*mU* G* A. 14136 575 287 mU. A. G. A.mC. A.mC. 288P.mU.fC. A.fU. A.fU. G.fU. 108% A.mU. A.mU. G. A.ChlG.fU.mC.mU. A*mC*mU* G*mU* G* G. 14137 671 289 mC. A. G. A.mC. G. A. 290P.mA.fU. G.fU.fC.fC.fU.fC.  98% G. G. A.mC. A.mU.ChlG.fU.mC.mU. G*mU* A* G*mC* A* U. 14138 924 291 mC. A. G.mC.mC. G.mU. 292P.mG. A. A.fU.fU.fC. A.fC. 100% G. A. A.mU.mU.mC.Chl G. G.mC.mU. G*A*mC*mU*mU*mU* G. 14139 1185 293 A. G.mU.mC.mU. G. G. 294P.mU.fU. A.fU.fU.fU.fC.fC.  47% A. A. A.mU. A. A.ChlA. G. A.mC.mU*mC* A* A* A*mU* A. 14140 1221 295 A. G.mU.mU.mU. 296P.mG. A. A. G.fC.fC. A.fC. 100% G.mU. G. A. A. A.mC.mU* A* A*G.mC.mU.mU.mC.Chl A*mC*mU* A. 14141 347 297 A. G.mU.mC.mC. A. 298P.mC.fU.fU.fU.fC. 103% A.mC. G. A. A. A. G.Chl G.fU.fU. G. G.A.mC.mU*mU* A*mC*mU*mU* G. 14142 634 299 A. A. G.mU.mU.mU.mC. 300P.mG.fU.fC.fU. G.fC. G. A. 100% G.mC. A. G. A.mC.Chl A.A.mC.mU.mU*mC*mU* mU* A* G* A. 14143 877 301 A. G.mC. A. A.mU. G. A. 302P.mA. A.fU. G.fC.fU.fC. 104% G.mC. A.mU.mU.Chl A.fU.fU.G.mC.mU*mC*mU*mC* A*mU* C. 14144 1033 303 mU.mU. A. G. A.mU. A. 304P.mA.fU. G.fC. A.fC.fU.  95% G.mU. G.mC. A.mU.Chl A.fU.fC.mU. A.A*mU*mU*mC* A*mU* G. 14145 714 305 mU. G. G.mU. G.mC. 306P.mC.fU.fU. G.fU. A.fU. 101% A.mU. A.mC. A. A. G.Chl G.fC. A.mC.mC.A*mU*mU*mC* A* A* C. 14146 791 307 A.mU. G. A. A. A.mC. G. 308P.mU. G. A.fC.fU.fC. 100% A. G.mU.mC. A.Chl G.fU.fU.fU.mC. A.mU* A*A*mC*mU* G* U. 14147 813 309 mC.mC. A. G. A. G.mU. 310P.mU.fU.fC. A. G.fC.  97% G.mC.mU. G. A. A.Chl A.fC.fU.fC.mU. G.G*mU*mC* A*mU*mC* C. 14148 939 311 mC. A. G.mC.mC. A.mU. 312P.mA. A. A.fU.fU.fC. A.fU. 109% G. A. A.mU.mU.mU.ChlG. G.mC.mU. G*mU* G* G* A* A* U. 14149 1161 313 A.mU.mU. G. 314P.mA.fC. A.fU.fU.fC. A.  34% G.mU.mU. G. A. A.mU.A.fC.fC. A. A.mU* A* A* G.mU.Chl A*mC*mU* G. 14150 1164 315G. G.mU.mU. G. A. 316 P.mU. A.fC. A.fC. n/a A.mU. G.mU. G.mU.A.fU.fU.fC. A. A.mC.mC* A.Chl A* A*mU* A* A* A. 14151 1190 317G. G. A. A. A.mU. A. 318 P.mA.fU.fU. A. G.fU.fU. n/aA.mC.mU. A. A.mU.Chl A.fU.fU.mU.mC.mC* A* G* A*mC*mU* C. 14152 1333 319mU.mC. A.mU. G. A. 320 P.mU.fU.fU.fC.fU.  31% A.mU. A. G. A. A. A.ChlA.fU.fU.fC. A.mU. G. A* G* A* G* A* A* U. 14153 537 321G.mC.mC. A. G.mC. A. 322 P.mU.fU.fC. G. G.fU.fU. n/aA.mC.mC. G. A. A.Chl G.fC.fU. G. G.mC* A* G* G*mU*mC* C. 14154 684 323mC. A.mC.mC.mU.mC. 324 P.mC. A.fU. G.fU. G.fU. G. 100% A.mC. A.mC. A.mU.A. G. G.mU. G* A*mU* G.Chl G*mU*mC* C. 14155 707 325 A. G.mU.mU. G. A.326 P.mG.fC. A.fC.fC.  99% A.mU. G. G.mU. A.fU.fU.fC. A. G.mC.ChlA.mC.mU*mC*mC*mU* mC* G* C. 14156 799 327 A. G.mU.mC. A. 328P.mC. A.fU.fC.fC. A.  95% G.mC.mU. G. G. A.mU. G.fC.fU. G. G.ChlA.mC.mU*mC* G*mU*mU*mU* C. 14157 853 329 mU. A.mU. A. A. G.mC. 330P.mC.fU.fU.fU.fC.fC. 106% G. G. A. A. A. G.Chl G.fC.fU.fU. A.mU. A*mU*A* A*mU*mC* U. 14158 888 331 mU.mU.mC.mC. G. 332 P.mA. A.fU.fC. A.fC. 88% A.mU. G.mU. G. A.fU.fC. G. G. A. A*mU* A.mU.mU.Chl G*mC*mU*mC* A.14159 1194 333 A.mU. A. A.mC.mU. A. 334 P.mA.fC. A.fC. A.fU.fU. A.  95%A.mU. G.mU. G.mU.Chl G.fU.mU. A.mU*mU*mU*mC*mC* A* G. 14160 1279 335mU.mC. 336 P.mU.fU.fC.fU. A.fU. A. G.  15% A.mU.mU.mC.mU.A. A.mU. G. A* A*mC* A.mU. A. G. A. A.Chl A*mU* A* G. 14161 1300 337A. A.mC.mU. A.mU.mC. 338 P.mU. A.fC. A. G.fU. G.  86%A.mC.mU. G.mU. A.Chl A.fU. A. G.mU.mU*mU* G*mC* A*mU* U. 14162 1510 339G.mU.mC. A. A.mU.mU. 340 P.mA.fU. A. A. G.fC. A.  86%G.mC.mU.mU. A.mU.Chl A.fU.fU. G. A.mC* A*mC*mC* A*mC* C. 14163 1543 341A. G.mC. A. A.mU.mU. 342 P.mU.fU.fU. A.fU.fU. A. 110%A. A.mU. A. A. A.Chl A.fU.fU. G.mC.mU* G* G* A*mC* A* A. 14164 434 343A.mC. G. 344 P.mU.fC. A.fU.fC. A. G. A. 134% A.mC.mU.mC.mU. G.G.fU.mC. G.mU*mU*mC* A.mU. G. A.Chl G* A* G* U. 14165 600 345mU. A. G.mU. G.mU. G. 346 P.mA.fU. A. A. A.fC.fC. 102% G.mU.mU.mU.A.fC. A.mC.mU. A.mU.Chl A*mU*mC* A*mC*mC* U. 14166 863 347A. A. G.mC.mC. A. 348 P.mU.fC. A.fU.fC. A.fU.fU.  93%A.mU. G. A.mU. G. A.Chl G. G.mC.mU.mU*mU*mC* mC* G*mC* U. 14167 902 349A.mU. A. G.mU.mC. A. 350 P.mA. G.fU.fU.fC.fC.fU. G. 101%G. G. A. A.mC.mU.Chl A.fC.mU. A.mU*mC* A* A*mU*mC* A. 14168 921 351A. G.mU.mC. A. 352 P.mU.fU.fC. A.fC. G.  98% G.mC.mC. G.mU. G. A.G.fC.fU. G. A.Chl A.mC.mU*mU*mU* G* G* A* A. 14169 154 353A.mC.mU. A.mC.mC. 354 P.mU.fU.fC.fU.fC. A.fU. G. n/aA.mU. G. A. G. A. A.Chl G.fU. A. G.mU* G* A* G*mU*mU* U. 14170 217 355A. A. A.mC. A. G. 356 P.mA. A.fU.fC. A.  66% G.mC.mU. G. G.fC.fC.fU.A.mU.mU.Chl G.mU.mU.mU* A* A*mC*mU* G* G. 14171 816 357G. A. G.mU. G.mC.mU. 358 P.mG. G.fU.fU.fU.fC. A. 102%G. A. A. A.mC.mC.Chl G.fC. A.mC.mU.mC*mU* G* G*mU*mC* A. 14172 882 359mU. G. A. G.mC. 360 P.mA.fU.fC. G. G. A. A.fU. 103% A.mU.mU.mC.mC. G.G.fC.mU.mC. A*mU*mU* A.mU.Chl G*mC*mU* C. 14173 932 361A. A.mU.mU.mC.mC. 362 P.mU. G. G.fC.fU. G.fU. G. n/a A.mC. A. G.mC.mC.G. A. A.mU.mU*mC* A.Chl A*mC* G* G* C. 14174 1509 363 mU. G.mU.mC. A.364 P.mU. A. A. G.fC. A. n/a A.mU.mU. A.fU.fU. G. A.mC.G.mC.mU.mU. A.Chl A*mC*mC* A*mC*mC* A. 14175 157 365A.mC.mC. A.mU. G. A. 366 P.mC. A. A.fU.fU.fC.fU.fC. 109%G. A. A.mU.mU. G.Chl A.fU. G. G.mU* A* G*mU* G* A* G. 14176 350 367mC.mC. A. A.mC. G. A. 368 P.mU. G. G.fC.fU.fU.fU.fC.  95%A. A. G.mC.mC. A.Chl G.fU.mU. G. G* A*mC*mU*mU* A* C. 14177 511 369mC.mU. G. G.mU.mC. 370 P.mA. A.fU.fC. A. G.fU. G. 100% A.mC.mU. G.A.fC.mC. A. A.mU.mU.Chl G*mU*mU*mC* A*mU* C. 14178 605 371mU. G. G.mU.mU.mU. 372 P.mA. G.fU.fC.fC. A.fU. A.  99% A.mU. G. G.A. A.mC.mC. A*mC* A.mC.mU.Chl A*mC*mU* A* U. 14179 811 373G. A.mC.mC. A. G. A. 374 P.mC. A. G.fC.  88% G.mU. G.mC.mU. G.ChlA.fC.fU.fC.fU. G. G.mU.mC* A*mU*mC*mC* A* G. 14180 892 375G. A.mU. G.mU. G. 376 P.mU. A.fU.fC. A. A.fU.fC.  76% A.mU.mU. G. A.mU.A.fC. A.mU.mC* G* G* A.Chl A* A*mU* G. 14181 922 377G.mU.mC. A. G.mC.mC. 378 P.mA.fU.fU.fC. A.fC. G.  59%G.mU. G. A. A.mU.Chl G.fC.fU. G. A.mC*mU*mU*mU* G* G* A. 14182 1169 379A. A.mU. G.mU. G.mU. 380 P.mA.fU. A. G. A.fU. A.fC.  69%A.mU.mC.mU. A.mU.Chl A.fC. A.mU.mU*mC* A* A*mC*mC* A. 14183 1182 381mU.mU. G. A. 382 P.mU.fU.fU.fC.fC. A. G. n/a G.mU.mC.mU. G. G. A.A.fC.fU.mC. A. A* A*mU* A. A.Chl A* G* A* U. 14184 1539 383G.mU.mC.mC. A. G.mC. 384 P.mU.fU. A. A.fU.fU.  77% A. A.mU.mU. A. A.ChlG.fC.fU. G. G. A.mC* A* A*mC*mC* G* U. 14185 1541 385 mC.mC. A. G.mC. A.386 P.mU. A.fU.fU. A. A.fU.fU. n/a A.mU.mU. A. A.mU.G.fC.mU. G. G* A*mC* A.Chl A* A*mC* C. 14186 427 387G. A.mC.mU.mC. G. A. 388 P.mA. G.fU.fC. G.fU.fU.fC.  69%A.mC. G. A.mC.mU.Chl G. A. G.mU.mC* A* A*mU* G* G* A. 14187 533 389A.mC.mC.mU. 390 P.mG.fU.fU. G.fC.fU. G.  78% G.mC.mC. A. G.mC. A.G.fC. A. G. A.mC.Chl G.mU*mC*mC* G*mU* G* G. 18538 496 391G. A.mU. G. A. 392 P.mU. A.fU.fC. A. G.  74% A.mU.mC.mU. G. A.mU.A.fU.fU.fC. A.fU.fC* A* A.Chl G* A* A*fU* G. 18539 496 393mU. G. A.mU. G. A. 394 P.mU. A.fU.fC. A. G.  72% A.mU.mC.mU. G. A.mU.A.fU.fU.fC. A.fU.fC* A* A.Chl G* A* A*fU* G. 18540 175 395 A.mU.mU.mU.396 P.mU. G.fC. A. A. A. A.  98% G.mC.mU.mU.mU.mU. G.fC. A. A. A.fU*fC*G.mC. A.Chl A*fC*fU*fG* C. 18541 175 397 G. A.mU.mU.mU. 398P.mU. G.fC. A. A. A. A.  28% G.mC.mU.mU.mU.mU. G.fC. A. A. A.fU*fC*G.mC. A.Chl A*fC*fU*fG* C. 18542 172 399 G.mU. G. 400P.mU. A. A. A. G.fC. A. A.  24% A.mU.mU.mU. A.fU.fC. A.fC*fU* G*fC*G.mC.mU.mU.mU. A.Chl A* A* U. 18543 172 401 A. G.mU. G. 402P.mU. A. A. A. G.fC. A. A.  14% A.mU.mU.mU. A.fU.fC. A.fC*fU* G*fC*G.mC.mU.mU.mU. A.Chl A* A* U. 18544 1013 403 A. A.mU.mU.mU.mC. 404P.mU. A. A. A.fU. A.fC. G. 100% G.mU. A.mU.mU.mU.A. A. A.fU.fU*fU*fC* A* A.Chl G* G* U. 18545 1013 405A. A. A.mU.mU.mU.mC. 406 P.mU. A. A. A.fU. A.fC. G. 109%G.mU. A.mU.mU.mU. A. A. A.fU.fU*fU*fC* A* A.Chl G* G* U. 18546 952 407mC. A.mC. A. G.mC.mC. 408 P.mU.fU.fU. C. A.fU. G.  32%A.mU. G. A. A. A.Chl G.fC.fU. G.fU. G* A* A* A*fU*fU* C. 18547 952 409mU.mC. A.mC. A. 410 P.mU.fU.fU. C. A.fU. G.  33% G.mC.mC. A.mU. G. A.G.fC.fU. G.fU. G* A* A* A. A.Chl A*fU*fU* C. 18548 174 411G. A.mU.mU.mU. 412 P.mU.fC. A. A. A. A. G.fC.  57% G.mC.mU.mU.mU.mU.A. A. A.fU.fC* A*fC*fU* G. A.Chl G*fC* A. 18549 174 413mU. G. A.mU.mU.mU. 414 P.mU.fC. A. A. A. A. G.fC.  53% G.mC.mU.mU.mU.mU.A. A. A.fU.fC* A*fC*fU* G. A.Chl G*fC* A. 18550 177 415 mU.mU. 416P.mU. A. G. G.fC. A. A. A.  97% G.mC.mU.mU.mU.mU.A. G.fC. A. A* A*fU*fC* G.mC.mC.mU. A.Chl A*fC* U. 18551 177 417mU.mU.mU. 418 P.mU. A. G. G.fC. A. A. A. 103% G.mC.mU.mU.mU.mU.A. G.fC. A. A* A*fU*fC* G.mC.mC.mU. A.Chl A*fC* U. 18552 1150 419mU.mU.mU.mC.mU.mC. 420 P.mU.fU. A. A. A.fC.fU. G.  96% A. G.mU.mU.mU. A.A. G. A. A. A* G* A* A* A.Chl G*fC* A. 18553 1089 421 mU.mU. G.mC. 422P.mU. G. A.fC.fU. A. A.  94% A.mU.mU.mU. A. A.fU. G.fC. A. A* A*G.mU.mC. A.Chl G*fU* G* A* G. 18554 1086 423 A.mC.mU.mU.mU. 424P.mU.fU. A. A. A.fU. G.fC. n/a G.mC. A.mU.mU.mU. A.A. A. A. G.fU* G* A* G* A.Chl A* A* A. 18555 1093 425 A.mU.mU.mU. A. 426P.mU.fU.fU.fU.fU. G. n/a G.mU.mC. A. A. A. A. A.fC.fU. A. A. A.fU* G*fC*A.Chl A* A* A* G. 18556 1147 427 mU.mU.mC.mU.mU.mU. 428P.mU. A.fC.fU. G. A. G. A. n/a mC.mU.mC. A. G.mU. A. A. G. A. A* G*fC*A.Chl A*fU*fU* U. 18557 1148 429 mU.mC.mU.mU.mU.mC. 430P.mU. A. A.fC.fU. G. A. G.  66% mU.mC. A. G.mU.mU.A. A. A. G. A* A* G*fC* A.Chl A*fU* U. 18558 1128 431G. A. A. A. G. A. G. A. 432 P.mU. A.fU.  16% A.mC. A.mU. A.ChlG.fU.fU.fC.fU.fC.fU.fU.fU. fC* A*fU*fU*fU*fU* G. 18559 1087 433mC.mU.mU.mU. G.mC. 434 P.mU.fC.fU. A. A. A.fU.  28% A.mU.mU.mU. A. G.G.fC. A. A. A. G*fU* G* A.Chl A* G* A* A. 18560 1088 435 mU.mU.mU. G.mC.436 P.mU. A.fC.fU. A. A. A.fU. n/a A.mU.mU.mU. A. G.mU.G.fC. A. A. A* G*fU* G* A.Chl A* G* A. 18561 1083 437 mC.mU.mC. 438P.mU. A.fU. G.fC. A. A. A.  53% A.mC.mU.mU.mU. G.fU. G. A. G* A* A*G.mC. A.mU. A.Chl A*fU*fU* G. 18562 1081 439 mU.mU.mC.mU.mC. 440P.mU. G.fC. A. A. A. G.fU.  89% A.mC.mU.mU.mU. G. A. G. A. A* A*fU*fU*G.mC. A.Chl G*fU* A. 18563 555 441 mC. A.mC.mU.mC.mC. 442P.mU. A.fC. A. A.fC.fU. G.  33% A. G.mU.mU. G.mU.G. A. G.fU. G* A* A* A* A.Chl A*fC*fU. 18564 1125 443A. A.mU. G. A. A. A. G. 444 P. mU.fU.fU.fC.fU.fC.fU.fU n/aA. G. A. A. A.Chl .fU.fC. A.fU.fU*fU*fU* G*fC*fU* A. 18565 168 445mU. G.mC. A. G.mU. G. 446 P.mU.fC. A. A. A.fU.fC.  14% A.mU.mU.mU.mG.A.fC.fU. G.fC. A* A.Chl A*fU*fU*fC*fU* C. 18566 1127 447mU. G. A. A. A. G. A. 448 P.mU.fU.  27% G. A. A.mC. A. A.ChlG.fU.fU.fC.fU.fC.fU.fU.fU. fC. A*fU*fU*fU*fU* G* C. 18567 1007 449A.mC.mC.mU. G. A. A. 450 P.mU. G. A. A. 129% A.mU.mU.mU.mC. A.ChlA.fU.fU.fU.fC. A. G. G.fU* G*fU*fU*fU* A* U. 18568 164 451G. A. A.mU.mU. G.mC. 452 P.mU.fU.fC. A.fC.fU. G.fC.  47%A. G.mU. G. A. A.Chl A. A.fU.fU.fC*fU*fC* A*fU* G* G. 18569 222 453G. G.mC.mU. G. 454 P.mU.fC.fC. A. G. A. n/a A.mU.mU.mC.mU. G. G.A.fU.fC. A. G.fC.fC*fU* A.Chl G*fU*fU*fU* A. 20612 172 455 A. G.mU. G.456 P.mU. A. A. A. G.fC. A. A. n/a A.mU.mU.mU. A.fU.mC. A.mC*mU*G.mC.mU.mU.mU. A.Chl G*mC* A* A* U. 20613 172 457 A. G.mU. G. 458P.mU. A. A. A. G.fC. A. A. n/a A.mU.mU.mU. A.fU.fC. A.mC*fU*G.mC.mU.mU.mU. A.Chl G*mC* A* A* U. 20614 172 459 A. G.mU. G. 460P.mU. A. A. A. G. C. A. A. 101% A.mU.mU.mU. A. U.mC. A.mC*mU*G.mC.mU.mU.mU. A.Chl G*mC* A* A* U. 20615 172 461 A. G.mU. G. 462P.mU. A. A. A. G.fC. A. A. 104% A.mU.mU.mU. A.fU.mC.G.mC.mU.mU.mU. A.Chl A.mC*mU*mG*mC*mA* mA* U. Key Chl = cholesterol withhydroxyprolinol linker TEG-chl = cholesterol with TEG linker m = 2′Ome f= 2′fluoro *= phosphorothioate llinkage .= phosphodiester linkage

TABLE 4  PTGS2 (Accession Number NM_000963.2) sd-rxRNA sequences OligoStart SEQ ID SEQ ID Number Site NO Sense sequence NO Antisense sequence% remaining expression (1 uM A549) 14422 451 463 mC. A.mC. 464P.mU.fC. A. A.fU.fC. A. 72% A.mU.mU.mU. G. A. A.fU. G.mU. G*A.mU.mU. G. A.Chl A*mU*mC*mU* G* G. 14423 1769 465 mC. A.mC.mU. 466P.mA. A.fU.fU. G. A. G. 71% G.mC.mC.mU.mC. G.fC. A. G.mU. A. A.mU.mU.ChlG*mU*mU* G* A*mU* G. 14424 1464 467 A. A. A.mU. 468P.mA. A. G. A.fC.fU. G. 74% A.mC.mC. A. G.fU. G.mU.mC.mU.mU.A.mU.mU.mU*mC* Chl A*mU*mC*mU* G. 14425 453 469 mC. A.mU.mU.mU. 470P.mU. G.fU.fC. A. 83% G. A.mU.mU. G. A.fU.fC. A. A. A.mU. A.mC. A.ChlG*mU* G* A*mU*mC* U. % remaining expression (1 uM PC-3) 17388 285 471G. A. A. A. 472 P.mU.fU. G. A. G.fC. A. 88% A.mC.mU.G.fU.fU.fU.fU.fC*fU*fC* G.mC.mU.mC. A. fC* A*fU* A. A.Chl 17389 520 473A.mC.mC.mU.mC. 474 P.mU. A. A.fU. A. G. G. 25% mU.mC.mC.mU.A. G. A. G. G.fU*fU* A* A.mU.mU. A.Chl G* A* G* A. 17390 467 475mU.mC.mC. 476 P.mU.fU. A. A. G.fU.fU. 68% A.mC.mC. A.G. G.fU. G. G. A*fC*fU* A.mC.mU.mU. A. G*fU*fC* A. A.Chl 17391 467 477G.mU.mC.mC. 478 P.mU.fU. A. A. G.fU.fU. 101% A.mC.mC. A.G. G.fU. G. G. A*fC*fU* A.mC.mU.mU. A. G*fU*fC* A. A.Chl   17392 524 479mC.mU.mC.mC.mU. 480 P.mU. G.fU. A.fU. A. 49% A.mU.mU. A.mU.A.fU. A. G. G. A. G* A* A.mC. A.Chl G* G*fU*fU* A. 17393 448 481G. A.mU.mC. 482 P.mU.fU.fC. A. A. A.fU. 29% A.mC.G.fU. G. A.fU.fC*fU* G* A.mU.mU.mU. G. G* A*fU* G. A. A.Chl 17394 448483 A. G. A.mU.mC. 484 P.mU.fU.fC. A. A. A.fU. 31% A.mC.G.fU. G. A.fU.fC*fU* G* A.mU.mU.mU. G. G* A*fU* G. A. A.Chl 17395 519485 A. 486 P.mU. A.fU. A. G. G. A. 12% A.mC.mC.mU.mC.G. A. G. G.fU.fU* A* G* mU.mC.mC.mU. A* G* A* A. A.mU. A.Chl 17396 437487 G.mU.mU. G. 488 P.mU.fC.fU. G. G. A.fU. 86% A.mC.G.fU.fC. A. A.fC* A*fC* A.mU.mC.mC. A. G. A*fU* A* A. A.Chl 17397 406489 mC.mC.mU.mU.mC. 490 P.mU.fU.fU.fC. G. A. A. 23% mC.mU.mU.mC. G.G. G. A. A. G. G* G* A* A. A. A.Chl A*fU* G* U. 17398 339 491A.mC.mU.mC.mC. 492 P.mU.fU. G.fU. 102%  A. A. A.mC. A.mC.G.fU.fU.fU. G. G. A. A. A.Chl G.fU* G* G* G*fU*fU* U. 17399 339 493 mC.494 P.mU.fU. G.fU. 55% A.mC.mU.mC.mC. G.fU.fU.fU. G. G. A.A. A. A.mC. A.mC. G.fU* G* G* G*fU*fU* A. A.Chl U. 17400 338 495 mC. 496P.mU. G.fU. G.fU.fU.fU. 62% A.mC.mU.mC.mC. G. G. A. G.fU. G* G*A. A. A.mC. A.mC. G*fU*fU*fU* C. A.Chl 17401 468 497 mC.mC. A.mC.mC. 498P.mU. G.fU. A. A. 61% A. A.mC.mU.mU. G.fU.fU. G. G.fU. G. G* A.mC. A.ChlA*fC*fU* G*fU* C. 17402 468 499 mU.mC.mC. 500 P.mU. G.fU. A. A. 179%A.mC.mC. A. G.fU.fU. G. G.fU. G. G* A.mC.mU.mU. A*fC*fU* G*fU* C.A.mC. A.Chl   174031  465 501 A. A.mU. A.mC.mC. 502P.mU. A. A. G. A.fC.fU. 30% A. G. G.fU. A.fU.fU*fU*fC* G.mU.mC.mU.mU.A*fU*fC* U. A.Chl 17404 243 503 G. A.mC.mC. A. 504 P.mU.fC.fU.fU. A.fU.32% G.mU. A.mU. A. A. A.fC.fU. G. G.fU.fC* A* G. A.Chl A* A*fU*fC* C.174051 472 505 G.mU.mC.mU.mU. 506 P.mU.fU.fC. A.fU.fU. A. 15%mU.mU. A. A.mU. A. A. A. G. A.fC*fU* G* G. A. A.Chl G*fU* A* U. 174062446 507 A. 508 P.mU. A. G. A.fC. A.fU. 142%  A.mU.mU.mU.mC.G. A. A. A.fU.fU* A.mU. A*fC*fU* G* G* U. G.mU.mC.mU. A.Chl 17407 449509 A.mU.mC. A.mC. 510 P.mU. A.fU.fC. A. A. 54% A.mU.mU.mU. G.A.fU. G.fU. G. A.mU. A.Chl A.fU*fC*fU* G* G* A* U. 17408 449 511G. A.mU.mC. 512 P.mU. A.fU.fC. A. A. 27% A.mC. A.fU. G.fU. G.A.mU.mU.mU. G. A.fU*fC*fU* G* G* A* A.mU. A.Chl U. 17409 444 513mU.mC.mC. A. G. 514 P.mU. A.fU. G.fU. G. 49% A.mU.mC. A.mC.A.fU.fC.fU. G. G. A*fU* A.mU. A.Chl G*fU*fC* A* A. 17410 1093 515mU. A.mC.mU. G. 516 P.mU.fC.fU.fC.fC.fU. 32% A.mU. A. G. G. A. G.A.fU.fC. A. G.fU. A.Chl A*fU*fU* A* G*fC* C. 17411 1134 517G.mU. G.mC. A. 518 P.mU.fC. A. A. G.fU. 70% A.mC. A.mC.mU.fU.G.fU.fU. G.mC. A.fC* G. A.Chl A*fU* A* A*fU* C. 17412 244 519A.mC.mC. A. 520 P.mU. A.fC.fU.fU. A.fU. 63% G.mU. A.mU. A. A.A.fC.fU. G. G.fU*fC* A* G.mU. A.Chl A* A*fU* C. 17413 1946 521 G. A. A.522 P.mU.fU.fC. A.fU.fU. A. 19% G.mU.mC.mU. A. G. A.fC.mU.fU.fC*fU*A.mU. G. A. A.Chl A*fC* A* G* U. 17414 638 523 A. A. G. A. A. G. A. 524P.mU. A. 27% A. A. G.mU.mU. A.fC.fU.fU.fU.fC.fU.fU.f A.ChlC.fU.fU* A* G* A* A* G* C. 17415 450 525 mU.mC. A.mC. 526P.mU. A. A.fU.fC. A. A. 216%  A.mU.mU.mU. G. A.fU. G.fU. G.A.mU.mU. A.Chl A*fU*fC*fU* G* G* A. 17416 450 527 A.mU.mC. A.mC. 528P.mU. A. A.fU.fC. A. A. 32% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU.mU. A.ChlA*fU*fC*fU* G* G* A. 17417 452 529 A.mC. 530 P.mU.fU.fC. A. A.fU.fC. 99%A.mU.mU.mU. G. A. A. A.fU. G.fU* G* A.mU.mU. G. A. A*fU*fC*fU* G. A.Chl17418 452 531 mC. A.mC. 532 P.mU.fU.fC. A. A.fU.fC. 54% A.mU.mU.mU. G.A. A. A.fU. G.fU* G* A.mU.mU. G. A. A*fU*fC*fU* G. A.Chl 17419 454 533A.mU.mU.mU. G. 534 P.mU.fU. G.fU.fC. A. 86% A.mU.mU. G. A.mC.A.fU.fC. A. A. A.fU* A. A.Chl G*fU* G* A*fU* C. 17420 454 535mC. A.mU.mU.mU. 536 P.mU.fU. G.fU.fC. A. 89% G. A.mU.mU. G.A.fU.fC. A. A. A.fU* A.mC. A. A.Chl G*fU* G* A*fU* C. 17421 1790 537mC. A.mU.mC.mU. 538 P.mU.fU.fU. A.fU.fU. 55% G.mC. A. A.mU. A.G.fC. A. G. A.fU. G* A* A. A.Chl G* A* G* A* C. 17422 1790 539 mU.mC.540 P.mU.fU.fU. A.fU.fU. 62% A.mU.mC.mU. G.fC. A. G. A.fU. G* A*G.mC. A. A.mU. A. G* A* G* A* C. A. A.Chl 21180 448 541 G. A.mU.mC. 542P.mU.fU.fC. A.mA. A.fU. 76% A.mC. G.fU. G. A.mU.mC*mU* A.mU.mU.mU. G.G* G* A*mU* G. A. A.TEG-Chl 21181 448 543 G. A.mU.mC. 544P.mU.fU.fC. A.mA. A.fU. 37% A.mC. G.fU. G. A.mU.mU.mU. G.A.fU.fC*fU*mG*mG*m A. A.TEG-Chl A*fU* G. 21182 448 545 G. A.mU.mC. 546P.mU.fU.fC. A. A. A.fU. 29% A.mC. G.fU. G. A.fU.fC*fU* G* A.mU.mU.mU.G* A*fU* G. G*mA*mA.TEG-Chl 21183 448 547 mG*mA*mU.mC. 548P.mU.fU.fC. A. A. A.fU. 46% A.mC. G.fU. G. A.fU.fC*fU* G* A.mU.mU.mU.G* A*fU* G. G*mA*mA.TEG-Chl 21184 448 549 mG*mA*mU.mC.m 550P.mU.fU.fC. A. A. A.fU. 60% A.mC.mA.mU.mU. G.fU. G. A.fU.fC*fU* G*mU.mG*mA*mA.T G* A*fU* G. EG-Chl 21185 449 551 G. A.mU.mC. 552P.mU. A.fU.fC. A. A. 27% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.fU.fC*fU* G* G* A.mU. A.TEG-Chl A*fU* G. 21186 449 553 G. A.mU.mC. 554P.mU. A.fU.fC. A. A. 57% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU.mC*mU* G* G* A.mU. A.TEG-Chl A*mU* G. 21187 449 555 G. A.mU.mC. 556P.mU. A.fU.fC. A.mA. 54% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU.mC*mU* G* G* A.mU. A.TEG-Chl A*mU* G. 21188 449 557 G. A.mU.mC. 558P.mU. A.fU.fC. A. A. 66% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU.mC*mU*mG*mG A.mU. A.TEG-Chl *mA*mU* G. 21189 449 559 G. A.mU.mC.560 P.mU. A.fU.fC. A.mA. 44% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU.mC*mU*mG*mG A.mU. A.TEG-Chl *mA*mU* G. 21190 449 561 G. A.mU.mC.562 P.mU. A.fU.fC. A. A. 52% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.fU.fC*fU*mG*mG*m A.mU. A.TEG-Chl A*fU* G. 21191 449 563 G. A.mU.mC.564 P.mU. A.fU.fC. A.mA. 41% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.fU.fC*fU*mG*mG*m A.mU. A.TEG-Chl A*fU* G. 21192 449 565 G. A.mU.mC.566 P.mU. A.fU.fC. A. A. 98% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.fU.mC*fU*mG*mG* A.mU. A.TEG-Chl mA*fU* G. 21193 449 567 G. A.mU.mC.568 P.mU. A.fU.fC. A. A. 93% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.fU*fC*fU* G* G* A* A*mU*mA.TEG-Chl U. 21194 449 569 mG*mA*mU.mC. 570P.mU. A.fU.fC. A. A. 119%  A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.fU*fC*fU* G* G* A* A*mU*mA.TEG-Chl U. 21195 449 571 mG*mA*mU.mC.m 572P.mU. A.fU.fC. A. A. 292%  A.mC.mA.mU.mU. A.fU. G.fU. G. mU.mG.mA*mU*mA.fU*fC*fU* G* G* A* A.TEG-Chl U. 20620 449 573 G. A.mU.mC. 574P.mU. A.fU.fC. A. A. 24% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU*mC*mU* G* G* A.mU. A.Chl-TEG A* U. 20621 449 575 G. A.mU.mC. 576P.mU. A.fU.fC. A. A.  5% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU*fC*mU* G* G* A.mU. A.Chl-TEG A* U. 20622 449 577 G. A.mU.mC. 578P.mU. A. U. C. A. A. A. 25% A.mC. U. G. U. G. A.mU.mU.mU. G.A.mU*mC*mU* G* G* A.mU. A.Chl-TEG A* U. 20623 449 579 G. A.mU.mC. 580P.mU. A.fU.fC. A. A. 14% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G.A.mU*mC*mU*mG*m A.mU. A.Chl-TEG G*mA* U. 20588 448 581 G. A.mU.mC. 582P.mU.fU.fC. A. A. A.fU. 17% A.mC. G.fU. G. A.mU.mC*mU* A.mU.mU.mU. G.G* G* A*mU* G. A. A.Chl-TEG 20589 448 583 G. A.mU.mC. 584P.mU.fU.fC. A. A. A.fU. 40% A.mC. G.fU. G. A.mU.fC*mU* A.mU.mU.mU. G.G* G* A*fU* G. A. A.Chl-TEG 20590 448 585 G. A.mU.mC. 586P.mU. U. C. A. A. A. U. 34% A.mC. G. U. G. A.mU.mC*mU* A.mU.mU.mU. G.G* G* A*mU* G. A. A.Chl-TEG 20591 448 587 G. A.mU.mC. 588P.mU.fU.fC. A. A. A.fU. n/a A.mC. G.fU. G. A.mU.mU.mU. G.A.fU.fC*fU*mG*mG*m A. A.Chl-TEG A*fU* G. Key Chl = cholesterol withhydroxyprolinol linker TEG-chl = cholesterol with TEG linker m = 2′Ome f= 2′fluoro *= phosphorothioate llinkage .= phosphodiester linkage

TABLE 5  CTGF (Accession Number: NM_001901.2) sd-rxRNA sequences% remaining mRNA expression Oligo Start SEQ ID SEQ ID (1 uM sd-rxRNA,Number Site NO Sense sequence NO Antisense sequence A549) 13980 1222 589A.mC. A. G. G. A. 590 P.mU. A.fC.  98% A. G. A.mU. G.mU.A.fU.fC.fU.fU.fC.fC.mU. A.Chl G.mU* A* G*mU* A*mC* A. 13981 813 591G. A. G.mU. G. G. 592 P.mA. G. G.fC.  82% A. G.mC. G.fC.fU.fC.fC.G.mC.mC.mU.Chl A.mC.mU.mC*mU* G*mU* G* G* U.   13982 747 593mC. G. A.mC.mU. 594 P.mU. 116% G. G. A. A. G. A.mC.G.fU.fC.fU.fU.fC.fC. A. A.Chl G.mU.mC. G* G*mU* A* A* G* C. 13983 817595 G. G. A. G.mC. 596 P.mG. A. A.fC. A. G.  97% G.mC.mC.mU.G.fC. G.fC.mU.mC.mC* G.mU.mU.mC.Chl A*mC*mU*mC*mU* G. 13984 1174 597G.mC.mC. 598 P.mC. A. G.fU.fU. G.fU. 102% A.mU.mU. A.mC. A.A. A.fU. G. G.mC* A* A.mC.mU. G.Chl G* G*mC* A* C. 13985 1005 599 G. A.600 P.mA. G.fC.fC. A. G. A. 114% G.mC.mU.mU.mU. A. A. G.mC.mU.mC* A*mC.mU. G. A* A*mC*mU* U. G.mC.mU.Chl 13986 814 601 A. G.mU. G. G. A. 602P.mC. A. G. G.fC. 111% G.mC. G.fC.fU.fC.fC. G.mC.mC.mU. A.mC.mU*mC*mU*G.Chl G*mU* G* G. 13987 816 603 mU. G. G. A. G.mC. 604P.mA. A.fC. A. G. G.fC. 102% G.mC.mC.mU. G.fC.fU.mC.mC. G.mU.mU.ChlA*mC*mU*mC*mU* G* U. 13988 1001 605 G.mU.mU.mU. G. 606 P.mA. G. A. A. A. 99% A. G.fC.fU.fC. A. A. G.mC.mU.mU.mU. A.mC*mU*mU* G* mC.mU.ChlA*mU* A. 13989 1173 607 mU. G.mC.mC. 608 P.mA. G.fU.fU. G.fU. A. 107%A.mU.mU. A.mC. A. A.fU. G. G.mC. A* G* A.mC.mU.Chl G*mC* A*mC* A. 13990749 609 A.mC.mU. G. G. A. 610 P.mC. G.fU.  91% A. G. A.mC. A.mC.G.fU.fC.fU.fU.fC.fC. A. G.Chl G.mU*mC* G* G*mU* A* A. 13991 792 611A. A.mC.mU. 612 P.mG. G. A.fC.fC. A. G.  97% G.mC.mC.mU. G.G.fC. A. G.mU.mU* G* G.mU.mC.mC.Chl G*mC*mU*mC* U. 13992 1162 613 A. G.614 P.mC. A. G. G.fC. A.fC. 107% A.mC.mC.mU. A. G. G.mU.G.mU.mC.mU*mU* G* G.mC.mC.mU. A*mU* G* A. G.Chl 13993 811 615mC. A. G. A. G.mU. 616 P.mG.fC. G.fC.fU.fC.fC. 113% G. G. A. G.mC.A.fC.fU.mC.mU. G*mU* G.mC.Chl G* G*mU*mC* U. 13994 797 617 mC.mC.mU. G.618 P.mG. G.fU.fC.fU. G. G. n/a G.mU.mC.mC. A. G.A.fC.fC. A. G. G*mC* A* A.mC.mC.Chl G*mU*mU* G. 13995 1175 619mC.mC. A.mU.mU. 620 P.mA.fC. A. G.fU.fU. 113% A.mC. A. A.mC.mU.G.fU. A. A.mU. G. G.mU.Chl G*mC* A* G* G*mC* A. 13996 1172 621mC.mU. G.mC.mC. 622 P.mG.fU.fU. G.fU. A. 110% A.mU.mU. A.mC. A.A.fU. G. G.mC. A. G* A.mC.Chl G*mC* A*mC* A* G. 13997 1177 623A.mU.mU. A.mC. 624 P.mG. G. A.fC. A. 105% A. A.mC.mU.G.fU.fU. G.fU. A. A.mU* G.mU.mC.mC.Chl G* G*mC* A* G* G. 13998 1176 625mC. A.mU.mU. 626 P.mG. A.fC. A. G.fU.fU.  89% A.mC. A. A.mC.mU.G.fU. A. A.mU. G* G.mU.mC.Chl G*mC* A* G* G* C. 13999 812 627A. G. A. G.mU. G. 628 P.mG. G.fC.  99% G. A. G.mC. G.fC.fU.fC.fC.G.mC.mC.Chl A.fC.mU.mC.mU* G*mU* G* G*mU* C. 14000 745 629 A.mC.mC. G.630 P.mU.fC.fU.fU.fC.fC. A. n/a A.mC.mU. G. G. A. G.fU.fC. G. G.mU* A*A. G. A.Chl A* G*mC*mC* G. 14001 1230 631 A.mU. G.mU. 632P.mU. G.fU.fC.fU.fC.fC. 106% A.mC. G. G. A. G. G.fU. A.mC. A.mC. A.ChlA.mU*mC*mU*mU*m C*mC* U. 14002 920 633 G.mC.mC.mU.mU. 634P.mA. G.fC.fU.fU.fC.  93% G.mC. G. A. A. G.fC. A. A. G. G.mC.mU.ChlG.mC*mC*mU* G* A*mC* C. 14003 679 635 G.mC.mU. G.mC. 636 P.mC. 102%G. A. G. G. A. A.fC.fU.fC.fC.fU.fC. G.mU. G.Chl G.fC. A. G.mC*A*mU*mU*mU*mC* C. 14004 992 637 G.mC.mC.mU. 638 P.mA. A. A.fC.fU.fU. G.100% A.mU.mC. A. A. A.fU. A. G. G.mU.mU.mU.Chl G.mC*mU*mU* G* G* A* G.14005 1045 639 A. 640 P.mA.fC.fU.fC.fC. A.fC. 104% A.mU.mU.mC.mU.A. G. A. A.mU.mU*mU* G.mU. G. G. A. A* G*mC*mU* C. G.mU.Chl 14006 1231641 mU. G.mU. A.mC. 642 P.mA.fU.  87% G. G. A. G. A.mC.G.fU.fC.fU.fC.fC. G.fU. A.mU.Chl A.mC. A*mU*mC*mU*mU*m C* C. 14007 991643 A. G.mC.mC.mU. 644 P.mA. A.fC.fU.fU. G. 101% A.mU.mC. A. A.A.fU. A. G. G.mU.mU.Chl G.mC.mU*mU* G* G* A* G* A. 14008 998 645mC. A. A. 646 P.mA. A. G.fC.fU.fC. A.  98% G.mU.mU.mU. G.A. A.fC.mU.mU. G* A. A*mU* A* G* G* C. G.mC.mU.mU.Chl 14009 1049 647mC.mU. G.mU. G. 648 P.mA.fC. A.fU.  98% G. A. G.mU. A.mU.A.fC.fU.fC.fC. A.mC. A. G.mU.Chl G* A* A*mU*mU*mU* A. 14010 1044 649A. A. 650 P.mC.fU.fC.fC. A.fC. A.  93% A.mU.mU.mC.mU.G. A. A.mU.mU.mU* A* G.mU. G. G. A. G*mC*mU*mC* G. G.Chl 14011 1327 651mU.mU.mU.mC. A. 652 P.mU. G.fU. G.fC.fU.  95% G.mU. A. G.mC.A.fC.fU. G. A. A. A.mC. A.Chl A*mU*mC* A*mU*mU* U. 14012 1196 653mC. A. A.mU. G. 654 P.mA. A. A. G. A.fU. 101% A.mC. G.fU.fC. A.mU.mU.A.mU.mC.mU.mU. G*mU*mC*mU*mC*m mU.Chl C* G. 14013 562 655 A. G.mU. 656P.mG.fU. G.fC. A.fC.fU.  66% A.mC.mC. A. G.mU. G. G.fU. G.mC. A.mC.ChlA.mC.mU*mU* G*mC* A* G* C. 14014 752 657 G. G. A. A. G. 658P.mA. A. A.fC. G.fU.  95% A.mC. A.mC. G.fU.fC.fU.mU.mC.mC*G.mU.mU.mU.Chl A* G*mU*mC* G* G. 14015 994 659 mC.mU. A.mU.mC. 660P.mU.fC. A. A.  85% A. A. A.fC.fU.fU. G. A.mU. A. G.mU.mU.mU. G.G* G*mC*mU*mU* G* A.Chl G. 14016 1040 661 A. G.mC.mU. A. A. 662P.mA.fC. A. G. A.  61% A.mU.mU.mC.mU. A.fU.fU.fU. A. G.mU.ChlG.mC.mU*mC* G* G*mU* A* U. 14017 1984 663 A. G. G.mU. A. G. 664P.mU.fU. A.fC.  32% A. A.mU. G.mU. A. A.fU.fU.fC.fU. A.ChlA.mC.mC.mU* A*mU* G* G*mU* G. 14018 2195 665 A. G.mC.mU. G. 666P.mA. A. A.fC.fU. G.  86% A.mU.mC. A. A.fU.fC. A. G.mC.mU*G.mU.mU.mU.Chl A*mU* A*mU* A* G. 14019 2043 667 mU.mU.mC.mU. 668P.mU. A.fU.fC.fU. G. A.  81% G.mC.mU.mC. A. G. G.fC. A. G. A.A.mU. A.Chl A*mU*mU*mU*mC*m C* A. 14020  1892 669 mU.mU. 670P.mU.fU. A. A.fC.fU.fU.  84% A.mU.mC.mU. A. A. A. G. A.mU. A.G.mU.mU. A. A.Chl A*mC*mU* G*mU* A* C. 14021 1567 671 mU. A.mU. A.mC.672 P.mU. A.fU.fU.  72% G. A. G.mU. A. A.fC.fU.fC. G.fU. A.mU.A.mU. A.Chl A* A* G* A*mU* G* C. 14022  1780 673 G. A.mC.mU. G. G. 674P.mA. A. G.fC.fU.  65% A.mC. A. G.fU.fC.fC. A. G.mC.mU.mU.ChlG.mU.mC*mU* A* A*mU*mC* G. 14023 2162 675 A.mU. G. 676P.mU. A. A.fU. A. A. A.  80% G.mC.mC.mU.mU. G. G.fC.mC. mU. A.mU.mU.A.mU*mU*mU* A.Chl G*mU*mU* C. 14024 1034 677 G.fC.fU.fC. G. G.mU. 678P.mU.fU.fU. A.  91% A.mU. A.mC.mC. A.mU* G. A. G.mC.mU. A.G*mU*mC*mU*mU* A. A.Chl C. 14025 2264 679 mU.mU. G.mU.mU. 680 P. mA.fC. 58% G. A. G. A. G.mU. A.fC.fU.fC.fU.fC. A. G.mU.ChlA.mC. A. A* A*mU* A* A* A* C. 14026 1032 681 A.mC. A.mU. 682P.mU. A. G.fC.fU.fC. G. 106% A.mC.mC. G. A. G.fU. A.mU. G.mC.mU. A.ChlG.mU*mC*mU*mU*m C* A* U. 14027 1535 683 A. G.mC. A. G. A. 684 P.mU. A. 67% A. A. G. G.mU.mU. A.fC.fC.fU.fU.fU.fC.fU. A.Chl G.mC.mU* G* G*mU*A*mC* C. 14028 1694 685 A. G.mU.mU. 686 P.mU.fU. A. A. G. G. A.  94%G.mU.mU.mC.mC. A.fC. A. A.mC.mU*mU* mU.mU. A. A.Chl G* A*mC*mU* C. 140291588 687 A.mU.mU.mU. G. 688 P.mU.fU. A.fC.  97% A. A. G.mU. G.mU.A.fC.fU.fU.fC. A. A. A. A.Chl A.mU* A* G*mC* A* G* G. 14030 928 689A. A. G.mC.mU. G. 690 P.mU.fC.fC. A. G. 100% A.mC.mC.mU. G. G.G.fU.fC. A. A.Chl G.mC.mU.mU*mC* G*mC* A* A* G. 14031 1133 691G. G.mU.mC. 692 P. mC.fU.fU.fC.fU.fU.fC.  82% A.mU. G. A. A. G. A.A.fU. G. A. G.Chl A.mC.mC*mU*mC* G*mC*mC* G. 14032  912 693 A.mU. G. 694P.mA. A. G. G.fC.fC.fU.  84% G.mU.mC. A. G. G. A.fC.mC. A.mU*G.mC.mC.mU.mU. G*mC* A*mC* A* G. Chl 14033 753 695 G. A. A. G. A.mC. 696P.mC. A. A. A.fC. G.fU.  86% A.mC. G.fU.fC.mU.mU.mC*m G.mU.mU.mU.C* A* G*mU*mC* G. G.Chl 14034 918 697 A. G. 698 P.mC.fU.fU.fC. G.fC. A. 88% G.mC.mC.mU.mU. A. G. G.mC.mC.mU* G* G.mC. G. A. A. A*mC*mC* A* U.G.Chl 14035 744 699 mU. A.mC.mC. G. 700 P.mC.fU.fU.fC.fC. A.  95%A.mC.mU. G. G. A. G.fU.fC. G. G.mU. A* A* A. G.Chl G*mC*mC* G* C. 14036466 701 A.mC.mC. G.mC. 702 P.mC.fC. G.  73% A. A. G. A.mU.mC.A.fU.fC.fU.fU. G.fC. G. G. G.Chl G.mU*mU* G* G*mC*mC* G. 14037 917 703mC. A. G. 704 P.mU.fU.fC. G.fC. A. A.  86% G.mC.mC.mU.mU.G. G.fC.mC.mU. G* G.mC. G. A. A.Chl A*mC*mC* A*mU* G. 14038  1038 705mC. G. A. 706 P.mA. G. A. A.fU.fU.fU.  84% G.mC.mU. A. A.A. G.fC.mU.mC. G* A.mU.mU.mC.mU. G*mU* A*mU* G* U. Chl 14039 1048 707mU.mC.mU. G.mU. 708 P.mC. A.fU.  87% G. G. A. G.mU.A.fC.fU.fC.fC. A.fC. A. G. A.mU. G.Chl A* A*mU*mU*mU* A* G. 14040 1235709 mC. G. G. A. G. 710 P.mU. G.fC.fC. A.fU. 100% A.mC. A.mU. G.G.fU.fC.fU.mC.mC. G.mC. A.Chl G*mU* A*mC* A*mU* C. 14041 868 711A.mU. G. A.mC. A. 712 P.mG. A. G. G.fC. 104% A.mC. G.fU.fU. G.fU.mC.G.mC.mC.mU.mC.Chl A.mU*mU* G* G*mU* A* A. 14042 1131 713G. A. G. G.mU.mC. 714 P.mU.fC.fU.fU.fC. A.fU.  85% A.mU. G. A. A. G.G. A.fC.mC.mU.mC* A.Chl G*mC*mC* G*mU* C. 14043 1043 715 mU. A. A. 716P.mU.fC.fC. A.fC. A. G.  74% A.mU.mU.mC.mU. A. A.fU.mU.mU. A*G.mU. G. G. A.Chl G*mC*mU*mC* G* G. 14044 751 717 mU. G. G. A. A. G. 718P.mA. A.fC. G.fU.  84% A.mC. A.mC. G.fU.fC.fU.fU.mC.mC. G.mU.mU.ChlA* G*mU*mC* G* G* U. 14045 1227 719 A. A. G. A.mU. 720P.mC.fU.fC.fC. G.fU.  99% G.mU. A.mC. G. G. A.fC. A. G.ChlA.fU.mC.mU.mU*mC* mC*mU* G*mU* A. 14046 867 721 A. A.mU. G. A.mC. 722P.mA. G. G.fC. G.fU.fU.  94% A. A.mC. G.fU.fC. A.mU.mU* G*G.mC.mC.mU.Chl G*mU* A* A* C. 14047 1128 723 G. G.mC. G. A. G. 724P.mU.fC. A.fU. G.  89% G.mU.mC. A.mU. A.fC.fC.fU.fC. G. A.ChlG.mC.mC* G*mU*mC* A* G* G. 14048 756 725 G. A.mC. A.mC. 726P.mG. G.fC.fC. A. A.  93% G.mU.mU.mU. G. A.fC. G.fU. G.mC.mC.ChlG.mU.mC*mU*mU*mC *mC* A* G. 14049 1234 727 A.mC. G. G. A. G. 728P.mG.fC.fC. A.fU. 100% A.mC. A.mU. G. G.fU.fC.fU.fC.mC. G.mC.ChlG.mU* A*mC* A*mU*mC* U. 14050 916 729 mU.mC. A. G. 730P.mU.fC. G.fC. A. A. G.  96% G.mC.mC.mU.mU. G.fC.fC.mU. G.G.mC. G. A.Chl A*mC*mC* A*mU* G* C. 14051 925 731 G.mC. G. A. A. 732P.mA. G. G.fU.fC. A.  80% G.mC.mU. G. G.fC.fU.fU.mC. G.mC*A.mC.mC.mU.Chl A* A* G* G*mC* C. 14052 1225 733 G. G. A. A. G. 734P.mC.fC. G.fU. A.fC.  96% A.mU. G.mU. A.fU.fC.fU.mU.mC.mC*A.mC. G. G.Chl mU* G*mU* A* G* U. 14053 445 735 G.mU. G. 736P.mG. A. G.fC.fC. G. A. 101% A.mC.mU.mU.mC. A. G.fU.mC. A.mC* A* G.G* A* A* G* A. G.mC.mU.mC.Chl 14054  446 737 mU. G. 738P.mG. G. A. G.fC.fC. G.  93% A.mC.mU.mU.mC. A. A. G.mU.mC. A*mC* G.A* G* A* A* G. G.mC.mU.mC.mC.Chl 14055 913 739 mU. G. G.mU.mC. 740P.mC. A. A. G.  67% A. G. G.fC.fC.fU. G. A.mC.mC. G.mC.mC.mU.mU.A*mU* G*mC* A*mC* G.Chl A. 14056 997 741 mU.mC. A. A. 742P.mA. G.fC.fU.fC. A. A.  92% G.mU.mU.mU. G. A.fC.fU.mU. G. A*mU*A. G.mC.mU.Chl A* G* G*mC* U. 14057 277 743 G.mC.mC. A. G. A. 744P.mC.fU. G.fC. A.  84% A.mC.mU. G.mC. A. G.fU.fU.fC.fU. G. G.ChlG.mC*mC* G* A*mC* G* G. 14058 1052 745 mU. G. G. A. G.mU. 746P.mG. G.fU. A.fC. A.fU. n/a A.mU. G.mU. A.fC.fU.mC.mC. A*mC* A.mC.mC.ChlA* G* A* A* U. 14059 887 747 G.mC.mU. A. G. A. 748 P. mC.fU.  80%G. A. A. G.mC. A. G.fC.fU.fU.fC.fU.fC.fU. G.Chl A. G.mC*mC*mU*G*mC* A* G. 14060 914 749 G. G.mU.mC. A. G. 750 P.mG.fC. A. A. G. 112%G.mC.mC.mU.mU. G.fC.fC.fU. G. G.mC.Chl A.mC.mC* A*mU* G*mC* A* C. 140611039 751 G. A. G.mC.mU. A. 752 P.mC. A. G. A. 104% A. A.fU.fU.fU. A.A.mU.mU.mC.mU. G.mC.mU.mC* G* G.Chl G*mU* A*mU* G. 14062 754 753A. A. G. A.mC. 754 P.mC.fC. A. A. A.fC. 109% A.mC. G.fU. G.mU.mU.mU. G.G.fU.mC.mU.mU*mC* G.Chl mC* A* G*mU* C. 14063 1130 755 mC. G. A. G. 756P.mC.fU.fU.fC. A.fU. G. 103% G.mU.mC. A.mU. A.fC.fC.mU.mC.G. A. A. G.Chl G*mC*mC* G*mU*mC* A. 14064 919 757 G. 758P.mG.fC.fU.fU.fC. G.fC. 109% G.mC.mC.mU.mU. A. A. G. G.mC.mC*mU*G.mC. G. A. A. G* A*mC*mC* A. G.mC.Chl 14065 922 759 mC.mU.mU. G.mC. 760P.mU.fC. A. 106% G. A. A. G.mC.mU. G.fC.fU.fU.fC. G.fC. A. G. A.ChlA. G* G*mC*mC*mU* G* A. 14066 746 761 mC.mC. G. 762P.mG.fU.fC.fU.fU.fC.fC. 106% A.mC.mU. G. G. A. A. G.fU.mC. G. G*mU*A. G. A.mC.Chl A* A* G*mC* C. 14067 993 763 mC.mC.mU. 764P.mC. A. A. A.fC.fU.fU.  67% A.mU.mC. A. A. G. A.fU. A. G. G.mU.mU.mU.G*mC*mU*mU* G* G* G.Chl A. 14068 825 765 mU. 766 P.mA. G. G.fU.fC.fU.fU. 93% G.mU.mU.mC.mC. G. G. A. A.mC. A* G* A. A. G. G*mC* G*mC* U.A.mC.mC.mU.Chl 14069 926 767 mC. G. A. A. 768 P.mC. A. G. G.fU.fC. A. 95% G.mC.mU. G. G.fC.fU.mU.mC. G*mC* A.mC.mC.mU. A* A* G* G* C. G.Chl14070 923 769 mU.mU. G.mC. G. 770 P.mG.fU.fC. A.  95% A. A. G.mC.mU. G.G.fC.fU.fU.fC. G.mC. A. A.mC.Chl A* G* G*mC*mC*mU* G. 14071 866 771mC. A. A.mU. G. 772 P.mG. G.fC. G.fU.fU. 132% A.mC. A. A.mC.G.fU.fC. A.mU.mU. G* G.mC.mC.Chl G*mU* A* A*mC* C. 14072 563 773G.mU. A.mC.mC. 774 P.mC. G.fU. G.fC. n/a A. G.mU. G.mC.A.fC.fU. G. G.mU. A.mC. G.Chl A.mC*mU*mU* G*mC* A* G. 14073 823 775mC.mC.mU. 776 P.mG.fU.fC.fU.fU. G. G.  98% G.mU.mU.mC.mC.A. A.fC. A. G. G*mC* A. A. G. A.mC.Chl G*mC*mU*mC* C. 14074 1233 777mU. A.mC. G. G. A. 778 P.mC.fC. A.fU. 109% G. A.mC. A.mU. G.G.fU.fC.fU.fC.fC. G.mU. G.Chl A*mC* A*mU*mC*mU* U. 14075 924 779mU. G.mC. G. A. A. 780 P.mG. G.fU.fC. A.  95% G.mC.mU. G.G.fC.fU.fU.fC. G.mC. A* A.mC.mC.Chl A* G* G*mC*mC* U. 14076 921 781mC.mC.mU.mU. 782 P.mC. A. G.fC.fU.fU.fC. 116% G.mC. G. A. A.G.fC. A. A. G. G.mC.mU. G.Chl G*mC*mC*mU* G* A* C. 14077 443 783mC.mU. G.mU. G. 784 P.mG.fC.fC. G. A. A. 110% A.mC.mU.mU.mC.G.fU.fC. A.mC. A. G* A* G. G.mC.Chl A* G* A* G* G. 14078 1041 785G.mC.mU. A. A. 786 P.mC. A.fC. A. G. A.  99% A.mU.mU.mC.mU.A.fU.fU.fU. A. G.mU. G.Chl G.mC*mU*mC* G* G*mU* A. 14079 1042 787mC.mU. A. A. 788 P.mC.fC. A.fC. A. G. A. 109% A.mU.mU.mC.mU.A.fU.fU.mU. A. G.mU. G. G.Chl G*mC*mU*mC* G* G* U. 14080 755 789A. G. A.mC. A.mC. 790 P.mG.fC.fC. A. A. A.fC. 121% G.mU.mU.mU. G. G.fU.G.mC.Chl G.mU.mC.mU*mU*mC *mC* A* G* U. 14081 467 791 mC.mC. G.mC. A.792 P.mG.fC. C.fG. A. 132% A. G. A.mU.mC. G. U.fC.fU.fU.fG. C.mG.G.mC.Chl G*mU*mU* G* G*mC* C. 14082 995 793 mU. A.mU.mC. A. 794P.mC.fU.fC. A. A. 105% A. G.mU.mU.mU. A.fC.fU.fU. G. A.mU. A*G. A. G.Chl G* G*mC*mU*mU* G. 14083 927 795 G. A. A. G.mC.mU. 796P.mC.fC. A. G. G.fU.fC. 114% G. A.mC.mC.mU. G. A. G.fC.mU.mU.mC* G.ChlG*mC* A* A* G* G. 17356 1267 797 A.mC. A.mU.mU. 798 P.mU. A.fU. G. A.120% A. A.mC.mU.mC. G.mU.fU. A. A.fU. A.mU. A.Chl G.fU*fC*fU*fC*fU*fC*A. 17357 1267 799 G. A.mC. 800 P.mU. A.fU. G. A.  56% A.mU.mU. A.G.mU.fU. A. A.fU. A.mC.mU.mC. G.fU*fC*fU*fC*fU*fC* A.mU. A.Chl A. 17358 1442 801 mU. G. A. A. G. A. 802 P.mU.fU. A. A.fC.  34% A.mU. G.mU.mU.A.fU.fU.fC.fU.fU.fC. A* A. A.Chl A* A*fC*fC* A* G. 17359 1442 803mU.mU. G. A. A. G. 804 P.mU.fU. A. A.fC.  31% A. A.mU.A.fU.fU.fC.fU.fU.fC. A* G.mU.mU. A. A.Chl A* A*fC*fC* A* G. 17360 1557805 G. A.mU. A. G.mC. 806 P.mU.fU. A. A. G. A.fU.  59% A.mU.mC.mU.mU.G.fC.fU. A.fU.fC*fU* G* A. A.Chl A*fU* G* A. 17361 1557 807A. G. A.mU. A. 808 P.mU.fU. A. A. G. A.fU.  47% G.mC.G.fC.fU. A.fU.fC*fU* G* A.mU.mC.mU.mU. A*fU* G* A. A. A.Chl 17362 1591809 mU. G. A. A. G.mU. 810 P.mU. A. A.fU.fU. A.fC. 120% G.mU. A.A.fC.fU.fU.fC. A* A* A.mU.mU. A.Chl A*fU* A* G* C. 17363 1599 811A. A.mU.mU. G. A. 812 P.mU.fU.fC.fC.fU.fU.fC.f  71% G. A. A. G. G. A.U.fC. A. A.fU.fU* A*fC* A.Chl A*fC*fU* U. 17364 1601 813mU.mU. G. A. G. A. 814 P.mU.fU.fU.fU.fC.fC.fU.  62% A. G. G. A. A. A.fU.fC.fU.fC. A. A.Chl A*fU*fU* A*fC* A* C. 17365 1732 815 mC. 816P.mU.fC. G. A. A.fU.fC.  99% A.mU.mU.mC.mU. A. G. A. A.fU. G*fU*fC*G. A.mU.mU.mC. A* G* A* G. G. A.Chl 17366 1734 817 mU.mU.mC.mU. G. 818P.mU.fU.fU.fC. G. A.  97% A.mU.mU.mC. G. A.fU.fC. A. G. A. A*fU*A. A. A.Chl G*fU*fC* A* G. 17367 1770 819 mC.mU. G.mU.mC. 820P.mU.fU.fC.fU. A.  45% G. A.mU.mU. A. G. A.fU.fC. G. A.fC. A. G*A. A.Chl G* A*fU*fU*fC* C. 17368 1805 821 mU.mU.mU. 822P.mU. G.fU.fU. A.fC. A.  71% G.mC.mC.mU. G. G.fC. A. A. G.mU. A. A.mC.A*fU*fU*fC* A*fC* U. A.Chl 17369 1805 823 A.mU.mU.mU. 824P.mU. G.fU.fU. A.fC. A.  67% G.mC.mC.mU. G. G.fC. A. A. G.mU. A. A.mC.A*fU*fU*fC* A*fC* U. A.Chl 17370 1815 825 A.mC. A. A. 826P.mU. A. A.fU.fC.fU. G.  65% G.mC.mC. A. G. G.fC.fU.fU. G.fU*fU*A.mU.mU. A.Chl A*fC* A* G* G. 17371 1815 827 A. A.mC. A. A. 828P.mU. A. A.fU.fC.fU. G.  35% G.mC.mC. A. G. G.fC.fU.fU. G.fU*fU*A.mU.mU. A.Chl A*fC* A* G* G. 173722 256 829 mC. A. 830P.mU. A.fC. A. A. A.fU. 113% G.mU.mU.mU. A. A. A.fC.fU. A.mU.mU.mU.G*fU*fC*fC* G* A* A. G.mU. A.Chl 173732 265 831 mU. G.mU.mU. G. 832P.mU. A.fC.  35% A. G. A. G.mU. A.fC.fU.fC.fU.fC. A. G.mU. A.ChlA.fC. A* A* A*fU* A* A* A. 173742 265 833 mU.mU. G.mU.mU. 834P.mU. A.fC.  31% G. A. G. A. G.mU. A.fC.fU.fC.fU.fC. A. G.mU. A.ChlA.fC. A* A* A*fU* A* A* A. 173752 295 835 mU. G.mC. 836P.mU.fU. A. G. A. A. A.  34% A.mC.mC.mU.mU. G. G.fU. G.fC. A* A*mU.mC.mU. A. A*fC* A*fU* G. A.Chl 17376 2295 837 mU.mU. G.mC. 838P.mU.fU. A. G. A. A. A.  28% A.mC.mC.mU.mU. G. G.fU. G.fC. A* A*mU.mC.mU. A. A*fC* A*fU* G. A.Chl 17377 1003 839 mU.mU. G. A. 840P.mU.fC. A. G. A. A. A.  67% G.mC.mU.mU.mU. G.fC.fU.fC. A. A*mC.mU. G. A.Chl A*fC*fU*fU* G* A. 17378 2268 841 mU. G. A. G. A. 842P.mU. G.fU.fC. A.fC.  42% G.mU. G.mU. G. A.fC.fU.fC.fU.fC. A*A.mC. A.Chl A*fC* A* A* A* U. 17379 2272 843 A. G.mU. G.mU. G. 844P.mU.fU.fU.fU. G.  35% A.mC.mC. A. A. A. G.fU.fC. A.fC. A.ChlA.fC.fU*fC*fU*fC* A* A* C. 17380 2272 845 G. A. G.mU. G.mU. 846P.mU.fU.fU.fU. G.  29% G. A.mC.mC. A. A. G.fU.fC. A.fC. A. A.ChlA.fC.fU*fC*fU*fC* A* A* C. 17381 2273 847 G.mU. G.mU. G. 848P.mU.fU.fU.fU.fU. G.  42% A.mC.mC. A. A. A. G.fU.fC. A.fC. A. A.ChlA.fC*fU*fC*fU*fC* A* A. 17382 2274 849 mU. G.mU. G. 850P.mU.fC.fU.fU.fU.fU. G.  42% A.mC.mC. A. A. A. G.fU.fC. A.fC.A. G. A.Chl A*fC*fU*fC*fU*fC* A. 17383 2274 851 G.mU. G.mU. G. 852P.mU.fC.fU.fU.fU.fU. G.  37% A.mC.mC. A. A. A. G.fU.fC. A.fC.A. G. A.Chl A*fC*fU*fC*fU*fC* A. 17384 2275 853 G.mU. G. 854P.mU. A.fC.fU.fU.fU.fU.  24% A.mC.mC. A. A. A. G. G.fU.fC. A.fC*A. G.mU. A.Chl A*fC*fU*fC*fU* C. 17385 2277 855 G. A.mC.mC. A. A. 856P.mU.fU. A.  27% A. A. G.mU.mU. A. A.fC.fU.fU.fU.fU. G. A.ChlG.fU.fC* A*fC* A*fC*fU* C. 17386 2296 857 G.mC. 858P.mU.fC.fU. A. G. A. A.  23% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* A*mU.mC.mU. A. G. A*fC* A* U. A.Chl 17387 2299 859 mC.mC.mU.mU. 860P.mU.fC. A. A.fC.fU. A.  46% mU.mC.mU. A. G. A. A. A. G. G*fU*G.mU.mU. G. A.Chl G*fC* A* A* A. 21138 2296 861 G.mC. 862P.mU.fC.fU. A. G. A.mA.  42% A.mC.mC.mU.mU. A. G. G.fU. G.mC* A*mU.mC.mU. A. G. A* A*mC* A* U. A.TEG-Chl 21139 2296 863 G.mC. 864P.mU.fC.fU. A. G.mA.  32% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC*mU.mC.mU. A. G. A* A* A*mC* A* U. A.TEG-Chl 21140 2296 865 G.mC. 866P.mU.fC.fU. A. G. A. A.  41% A.mC.mC.mU.mU. A. G. G.fU. G.mC*mU.mC.mU. A. G. A*mA* A*mC* A* U. A.TEG-Chl 21141 2296 867 G.mC. 868P.mU.fC.fU. A. G. A.mA.  51% A.mC.mC.mU.mU. A. G. G.fU. G.mC*mU.mC.mU. A. G. A*mA* A*mC* A* U. A.TEG-Chl 21142 2296 869 G.mC. 870P.mU.fC.fU. A. G.mA.  25% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC*mU.mC.mU. A. G. A*mA* A*mC* A* U. A.TEG-Chl 21143 2296 871 G.mC. 872P.mU.fC.fU. A. G. A. A.  61% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU. A. G.G.fC*mA*mA*mA*fC* A.TEG-Chl mA* U. 21144 2296 873 G.mC. 874P.mU.fC.fU. A. G. A.mA.  49% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU. A. G.G.fC*mA*mA*mA*fC* A.TEG-Chl mA* U. 21145 2296 875 G.mC. 876P.mU.fC.fU. A. G.mA.  46% A.mC.mC.mU.mU. A.mA. G. G.fU. mU.mC.mU. A. G.G.fC*mA*mA*mA*fC* A.TEG-Chl mA* U. 21146 2296 877 G.mC. 878P.mU.fC.fU. A. G. A. A.  37% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* A*mU.mC.mU. A*fC* A* U. A*mG*mA.TEG-Chl 21147 2296 879 mG*mC* 880P.mU.fC.fU. A. G. A. A.  43% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* A*mU.mC.mU. A*fC* A* U. A*mG*mA.TEG-Chl 21148 2296 881 mG*mC*mA.mC. 882P.mU.fC.fU. A. G. A. A.  29% mC.mU.mU.mU.mC. A. G. G.fU. G.fC* A* A*mU.mA*mG*mA. A*fC* A* U. TEG-Chl 21149 2275 883 G.mU. G. 884P.mU. A.fC.fU.fU.fU.fU. 138% A.mC.mC. A. A. A. G. G.fU.fC. A.fC*A. G*mU*mA.TEG- A*fC*fU*fC*fU* C. Chl 21150 2275 885 mG*mU* G. 886P.mU. A.fC.fU.fU.fU.fU. 116% A.mC.mC. A. A.mA. G. G.fU.fC. A.fC*A. G*mU*mA.TEG- A*fC*fU*fC*fU* C. Chl 21151 2275 887 mG*mU*mG.mA. 888P.mU. A.fC.fU.fU.fU.fU. 105% mC.mC.mA.mA.mA G. G.fU.fC. A.fC*.mA.mG*mU*mA.T A*fC*fU*fC*fU* C. EG-Chl 21152  2295 889 mU.mU. G.mC. 890P.mU.fU. A. G. A.mA. A.  46% A.mC.mC.mU.mU. G. G.fU. G.fC. A. A*mU.mC.mU. A. A*fC* A*fA* G* G. A.TEG-Chl 21153 2295 891 mU.mU. G.mC. 892P.mU.fU. A. G.mA.  28% A.mC.mC.mU.mU. A.mA. G. G.fU. G.fC. A.mU.mC.mU. A. A* A*fC* A*fA* G* G. A.TEG-Chl 21154 2295 893 mU.mU. G.mC.894 P.mU.fU.mA. G.mA.  28% A.mC.mC.mU.mU. A.mA. G.mG.fU. G.fC.mU.mC.mU. A. A. A* A*fC* A*fA* G* A.TEG-Chl G. 21155 2295 895mU.mU. G.mC. 896 P.mU.fU. A. G. A.mA. A.  60% A.mC.mC.mU.mU.G. G.fU. G.mC. A. A* mU.mC.mU. A. A*mC* A*mA* G* G. A.TEG-Chl 21156 2295897 mU.mU. G.mC. 898 P.mU.fU. A. G. A.mA. A.  54% A.mC.mC.mU.mU.G. G.fU. G.fC. mU.mC.mU. A. A.mA*mA*fC*mA*fA* A.TEG-Chl mG* G. 211572295 899 mU.mU. G.mC. 900 P.mU.fU. A. G. A.mA. A.  40% A.mC.mC.mU.mU.G. G.fU. mU.mC.mU. A. G.fC.mA.mA*mA*fC*m A.TEG-Chl A*fA*mG* G. 211582295 901 mU.mU. G.mC. 902 P.mU.fU. A. G. A.mA. A. n/a A.mC.mC.mU.mU.G. G.fU. G.fC. mU.mC.mU. A. A.mA*mA*fC*mA*mA* A.TEG-Chl mG* G. 211592295 903 mU.mU. G.mC. 904 P.mU.fU. A. G. A.mA. A.  41% A.mC.mC.mU.mU.G. G.fU. G.fC. mU.mC.mU. A. A.mA*mA*mC*mA*mA A.TEG-Chl *mG* G. 211602295 905 mU.mU. G.mC. 906 P.mU.fU. A. G. A.mA. A.  65% A.mC.mC.mU.mU.G. G.fU. G.fC.mA. mU.mC.mU. A. A*mA*mC*mA*mA*m A.Chl-TEG G*mG. 211612295 907 mU.mU. G.mC. 908 P.mU.fU. A. G. A.mA. A.  43% A.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU. A. A*fC* A*mA*mG* G. A.TEG-Chl 21162 2295909 mU.mU. G.mC. 910 P.mU.fU. A. G. A.mA. A.  41% A.mC.mC.mU.mU.G. G.fU. G.fC.mA. mU.mC.mU. A. A*mA*fC* A*mA*mG* A.TEG-Chl G. 21163 2295911 mU.mU. G.mC. 912 P.mU.fU. A. G. A. A. A.  32% A.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU. A* A*fC* A* A* G* G. A*TEG-Chl 21164 2295913 mU.mU. G.mC. 914 P.mU.fU. A. G. A. A. A.  39% A.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU.mA* A*fC* A* A* G* G. mA*TEG-Chl 211652295 915 mU*mU* G.mC. 916 P.mU.fU. A. G. A. A. A.  28% A.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU.mA* A*fC* A* A* G* G. mA*TEG-Chl 211662295 917 mU.mU.mG.mC. 918 P.mU.fU. A. G. A. A. A.  27% mA.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU.mA* A*fC* A* A* G* G. mA*TEG-Chl 211672299 919 mC.mC.mU.mU. 920 P.mU.fC. A. A.fC.fU. A.  49% mU.mC.mU. A.G. A.mA. A. G. G*fU* G.mU.mU. G. G*fC* A* A* A. A.TEG-Chl 21168 2299 921mC.mC.mU.mU. 922 P.mU.fC. A. A.fC.fU. A.  53% mU.mC.mU. A.G. A.mA. A. G. G*mU* G.mU.mU. G. G*mC* A* A* A. A.TEG-Chl 21169 2299 923mC.mC.mU.mU. 924 P.mU.fC. A. A.fC.fU. A.  47% mU.mC.mU. A.G.mA. A. A.mG. G*fU* G.mU.mU. G. G*fC* A* A* A. A.TEG-Chl 21170 2299 925mC.mC.mU.mU. 926 P.mU.fC. A. A.fC.fU. A.  70% mU.mC.mU. A.G.mA. A. A.mG. G*mU* G.mU.mU. G. G*mC* A* A* A. A.TEG-Chl 21171 2299 927mC.mC.mU.mU. 928 P.mU.fC. A. A.fC.fU. A.  65% mU.mC.mU. A.G. A.mA. A. G. G*mU* G.mU.mU. G. G*mC* A*mA* A. A.TEG-Chl 21172 2299 929mC.mC.mU.mU. 930 P.mU.fC. A. A.fC.fU. A.  43% mU.mC.mU. A.G. A.mA. A. G. G*mU* G.mU.mU. G. G*mC*mA*mA* A. A.TEG-Chl 21173 2299 931mC.mC.mU.mU. 932 P.mU.fC. A. A.fC.fU. A.  52% mU.mC.mU. A. G. A.mA. A.G.mU.mU. G. G.mG*mU*mG*mC*m A.TEG-Chl A*mA* A. 21174 2299 933mC.mC.mU.mU. 934 P.mU.fC. A. A.fC.fU. A.  47% mU.mC.mU. A.G. A.mA. A. G. G.mU.mU. G. G*mU*mG*mC*mA*m A.TEG-Chl A* A. 21175 2299935 mC.mC.mU.mU. 936 P.mU.fC. A. A.fC.fU. A.  35% mU.mC.mU. A.G. A.mA. A. G. G.mU.mU. G. G*fU*mG*fC*mA*mA* A.TEG-Chl A. 21176 2299 937mC.mC.mU.mU. 938 P.mU.fC. A. A.fC.fU. A.  50% mU.mC.mU. A.G.mA. A. A.mG. G.mU.mU. G. G*fU*mG*fC*mA*mA* A.TEG-Chl A. 21177 2299 939mC.mC.mU.mU. 940 P.mU.fC. A. A.fC.fU. A.  37% mU.mC.mU. A.G. A. A. A. G. G*fU* G.mU.mU*mG*mA G*fC* A* A* A. .TEG-Chl 21178 2299941 mC*mC*mU.mU. 942 P.mU.fC. A. A.fC.fU. A.  36% mU.mC.mU. A.G. A. A. A. G. G*fU* G.mU.mU*mG*mA. G*fC* A* A* A. TEG-Chl 21179 2299943 mC*mC*mU.mU. 944 P.mU.fC. A. A.fC.fU. A.  35% mU.mC.mU.mA.mG.G. A. A. A. G. G*fU* mU.mU*mG*mA. G*fC* A* A* A. TEG-Chl 21203 2296 945G.mC. 946 P.mU.fC.fU. A. G. A.mA.  40% A.mC.mC.mU.mU.A. G. G.fU. G.mC* A* mU.mC.mU. A* A*mC* A* U. A*mG*mA.TEG-Chl 21204 2296947 G.mC. 948 P.mU.fC.fU. A. G.mA.  28% A.mC.mC.mU.mU.A.mA. G. G.fU. G.mC* mU.mC.mU. A* A* A*mC* A* U. A*mG*mA.TEG-Chl 212052296 949 G.mC. 950 P.mU.fC.fU. A. G.mA.  51% A.mC.mC.mU.mU.A.mA. G. G.fU. G.mC* mU.mC.mU. A*mA* A*mC* A* U. A*mG*mA.TEG-Chl  212062296 951 mG*mC* 952 P.mU.fC.fU. A. G. A.mA.  46% A.mC.mC.mU.mU.A. G. G.fU. G.mC* A* mU.mC.mU. A* A*mC* A* U. A*mG*mA.TEG-Chl  212072296 953 mG*mC* 954 P.mU.fC.fU. A. G.mA.  29% A.mC.mC.mU.mU.A.mA. G. G.fU. G.mC* mU.mC.mU. A* A* A*mC* A* U. A*mG*mA.TEG-Chl  212082296 955 mG*mC* 956 P.mU.fC.fU. A. G.mA.  72% A.mC.mC.mU.mU.A.mA. G. G.fU. G.mC* mU.mC.mU. A*mA* A*mC* A* U. A*mG*mA.TEG-Chl  212092296 957 mG*mC*mA.mC. 958 P.mU.fC.fU. A. G. A.mA.  89% mC.mU.mU.mU.mC.A. G. G.fU. G.mC* A* mU.mA*mG*mA. A* A*mC* A* U. TEG-Chl 21210 2296 959mG*mC*mA.mC. 960 P.mU.fC.fU. A. G.mA.  65% mC.mU.mU.mU.mC.A.mA. G. G.fU. G.mC* mU.mA*mG*mA. A* A* A*mC* A* U. TEG-Chl 21211 2296961 mG*mC*mA.mC. 962 P.mU.fC.fU. A. G.mA.  90% mC.mU.mU.mU.mC.A.mA. G. G.fU. G.mC* mU.mA*mG*mA. A*mA* A*mC* A* U. TEG-Chl 21212 2295963 mU.mU. G.mC. 964 P.mU.fU. A. G. A.mA. A.  60% A.mC.mC.mU.mU.G. G.fU. mU.mC.mU*mA* G.fC.mA.mA*mA*fC*m mA.TEG-Chl A*mA*mG* G. 212132295 965 mU.mU. G.mC. 966 P.mU.fU. A. G. A.mA. A.  63% A.mC.mC.mU.mU.G. G.fU. G.fC. mU.mC.mU*mA* A.mA*mA*mC*mA*mA mA.TEG-Chl *mG* G. 212142295 967 mU.mU. G.mC. 968 P.mU.fU. A. G. A.mA. A.  52% A.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU*mA* A*fC* A*mA*mG* G. mA.TEG-Chl 212152295 969 mU.mU. G.mC. 970 P.mU.fU. A. G. A.mA. A.  45% A.mC.mC.mU.mU.G. G.fU. G.fC.mA. mU.mC.mU*mA* A*mA*fC* A*mA*mG* mA.TEG-Chl G. 212162295 971 mU*mU* G.mC. 972 P.mU.fU. A. G. A.mA. A.  65% A.mC.mC.mU.mU.G. G.fU. mU.mC.mU*mA*m G.fC.mA.mA*mA*fC*m A.TEG-Chl A*mA*mG* G. 212172295 973 mU*mU* G.mC. 974 P.mU.fU. A. G. A.mA. A.  69% A.mC.mC.mU.mU.G. G.fU. G.fC. mU.mC.mU*mA* A.mA*mA*mC*mA*mA mA.TEG-Chl *mG* G. 212182295 975 mU*mU* G.mC. 976 P.mU.fU. A. G. A.mA. A.  62% A.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU*mA* A*fC* A*mA*mG* G. mA.TEG-Chl 212192295 977 mU*mU* G.mC. 978 P.mU.fU. A. G. A.mA. A.  54% A.mC.mC.mU.mU.G. G.fU. G.fC.mA. mU.mC.mU*mA* A*mA*fC* A*mA*mG* mA.TEG-Chl G. 212202295 979 mU.mU.mG.mC. 980 P.mU.fU. A. G. A.mA. A.  52% mA.mC.mC.mU.mU.G. G.fU. mU.mC.mU*mA* G.fC.mA.mA*mA*fC*m mA.TEG-Chl A*mA*mG* G. 212212295 981 mU.mU.mG.mC. 982 P.mU.fU. A. G. A.mA. A.  53% mA.mC.mC.mU.mU.G. G.fU. G.fC. mU.mC.mU*mA* A.mA*mA*mC*mA*mA mA.TEG-Chl *mG* G. 212222295 983 mU.mU.mG.mC. 984 P.mU.fU. A. G. A.mA. A.  43% mA.mC.mC.mU.mU.G. G.fU. G.fC. A. A* mU.mC.mU*mA* A*fC* A*mA*mG* G. mA.TEG-Chl 212232295 985 mU.mU.mG.mC. 986 P.mU.fU. A. G. A.mA. A.  43% mA.mC.mC.mU.mU.G. G.fU. G.fC.mA. mU.mC.mU*mA* A*mA*fC* A*mA*mG* mA.TEG-Chl G. 212242299 987 mC.mC.mU.mU. 988 P.mU.fC. A. A.fC.fU. A.  60% mU.mC.mU. A.G. A.mA. A. G. G.mU.mU*mG*mA. G*fU*mG*fC*mA*mA* TEG-Chl A. 21225 2299989 mC*mC*mU.mU. 990 P.mU.fC. A. A.fC.fU. A.  67% mU.mC.mU. A.G. A.mA. A. G. G.mU.mU*mG*mA. G*fU*mG*fC*mA*mA* TEG-Chl A. 21226 2299991 mC*mC*mU.mU. 992 P.mU.fC. A. A.fC.fU. A.  66% mU.mC.mU.mA.mG.G. A.mA. A. G. mU.mU*mG*mA.T G*fU*mG*fC*mA*mA* EG-Chl A. 21227 2296 993G.mC. 994 P.mU.fC.fU. A. G.mA.  49% A.mC.mC.mU.mU. A.mA. G. G.fU.mU.mC.mU. G.fC*mA*mA*mA*fC* A*mG*mA.TEG-Chl mA* U. 20584 2296 995 G.mC.996 P.mU.fC.fU. A. G. A. A.  70% A.mC.mC.mU.mU. A. G. G.mU. G.mC* A*mU.mC.mU. A. G. A* A*mC* A* U. A.Chl-TEG 20585 2296 997 G.mC. 998P.mU.fC.fU. A. G. A. A.  15% A.mC.mC.mU.mU. A. G. G.fU. G.mC* A*mU.mC.mU. A. G. A* A*mC* A* U. A.Chl-TEG 20586 2296 999 G.mC. 1000P.mU. C. U. A. G. A. A.  30% A.mC.mC.mU.mU. A. G. G.mU. G.mC* A*mU.mC.mU. A. G. A* A*mC* A* U. A.Chl-TEG 20587 2296 1001 G.mC. 1002P.mU.fC.fU. A. G. A. A.  32% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU. A. G.G.fC*mA*mA*mA*fC* A.Chl-TEG mA* U. 20616 2275 1003 G.mU. G. 1004P.mU. A.fC.fU.fU.fU.fU.  22% A.mC.mC. A. A. A. G. G.fU.mC. A.mC*A. G.mU. A.Chl-TEG A*mC*mU*mC*mU* C. 20617 2275 1005 G.mU. G. 1006P.mU. A.fC.fU.fU.fU.fU.  18% A.mC.mC. A. A. A. G. G.fU.fC. A.mC*A. G.mU. A.Chl-TEG A*fC*mU*fC*mU* C. 20618 2275 1007 G.mU. G. 1008P.mU. A. C. U. U. U. U.  36% A.mC.mC. A. A. A. G. G. U.mC. A.mC*A. G.mU. A.Chl-TEG A*mC*mU*mC*mU* C. 20619 2275 1009 G.mU. G. 1010P.mU. A.fC.fU.fU.fU.fU.  28% A.mC.mC. A. A. A. G. G.fU.fC.A. G.mU. A.Chl-TEG A.mC*mA*mC*mU*mC *mU* C. 21381 2275 1011 G.mU. G.1012 P.mU. A.fC.fU.fU.fU.fU.  28% A.mC.mC. A. A. A. G. G.fU.mC. A.mC*A. G*mU*mA.TEG- A*mC*mU*mC*mU* C. Chl 21382 2275 1013 G.mU. G. 1014P.mU. A.fC.fU.fU.fU.fU.  28% A.mC.mC. A. A. A. G. G.fU.fC. A.mC*A. G*mU*mA.TEG- A*fC*mU*fC*mU* C. Chl 21383 2275 1015 mG*mU*mG.mA. 1016P.mU. A.fC.fU.fU.fU.fU.  43% mC.mC.mA.mA.mA. G. G.fU.mC. A.mC*mA.mG*mU*mA. A*mC*mU*mC*mU* C. TEG-Chl 21384 2275 1017 mG*mU*mG.mA. 1018P.mU. A.fC.fU.fU.fU.fU.  50% mC.mC.mA.mA.mA. G. G.fU.fC. A.mC*mA.mG*mU*mA. A*fC*mU*fC*mU* C. TEG-Chl 20392 2275 1019 G.mU. G. 1020P.mU. A.fC.fU.fU.fU.fU.  28% A.mC.mC. A. A. A. G. G.fU.fC. A.fC*A. G.mU. A.TEG-Chl A*fC*fU*fC*fU* C. 20393 2296 1021 G.mC. 1022P.mU.fC.fU. A. G. A. A.  35% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* A*mU.mC.mU. A. G. A*fC* A* U. A.TEG-Chl 21429 2275 1023 G.mU. G. 1024P.mU. A.fC.fU.fU.fU.fU.  36% A.mC.mC. A. A. A. G. G.fU.fC. A.mC*A. G*mU*mA.Teg- A*fC*mU*fC*mU* C. Chl 21430 2275 1025 G.mU. G. 1026P.mU. A.fC.fU.fU.fU.fU.  31% A.mC.mC. A. A.mA. G. G.fU.mC. A.mC*A. G*mU*mA.Teg- A*mC*mU*mC*mU* C. Chl Key Chl = cholesterol withhydroxyprolinol linker TEG-chl = cholesterol with TEG linker m = 2′Ome f= 2′fluoro *= phosphorothioate llinkage .= phosphodiester linkage

TABLE 6  TGFβ2 (Accession Number: NM_001135599.1) sd-rxRNA sequences% remaining Oligo Start SEQ ID SEQ ID expression Number Site NOSense sequence NO Antisense sequence (1 uM, A549) 14408 1324 1027 G.1028 P.mU.fC. G. A. A. G. G.  94% G.mC.mU.mC.mU. A. G. A. G.mC.mC*mC.mC.mU.mU.mC. A*mU*mU*mC* G* C. G. A.Chl 14409 1374 1029G. A.mC. A. G. G. 1030 P.mC.fC. A. G. n/a A. A.mC.mC.mU. G.G.fU.fU.fC.fC.fU. G.Chl G.mU.mC*mU*mU*m U* A*mU* G. 14410 946 1031mC.mC. A. A. G. G. 1032 P.mU. A. A.  90% A. G. A.fC.fC.fU.fC.fC.fU.mU.G.mU.mU.mU. G. G*mC* G*mU* A* A.Chl G* U. 14411 849 1033 A.mU.mU.mU.mC.1034 P.mU. G.fU. A. G. A.fU.  72% mC. A.mU.mC.mU. G. G. A. A. A.mU*mC*A.mC. A.Chl A*mC*mC*mU* C. 14412 852 1035 mU.mC.mC. 1036P.mU. G.fU.fU. G.fU. A.  76% A.mU.mC.mU. G. A.fU. G. G. A* A*A.mC. A. A.mC. A*mU*mC* A* C. A.Chl   14413 850 1037 mU.mU.mU.mC.m 1038P.mU.fU. G.fU. A. G.  98% C. A.mU.mC.mU. A.fU. G. G. A. A.A.mC. A. A.Chl A*mU*mC* A*mC*mC* U.   14414 944 1039 mC. G.mC.mC. A.1040 P.mA. 100% A. G. G. A. G. A.fC.fC.fU.fC.fC.fU.fU. G.mU.mU.ChlG. G.mC. G*mU* A* G*mU* A* C. 14415 1513 1041 G.mU. G. G.mU. G. 1042P.mU.fU.fC.fU. G. n/a A.mU.mC. A. G. A. A.fU.fC. A.fC.mC. A.ChlA.mC*mU* G* G*mU* A* U. 14416 1572 1043 mC.mU.mC.mC.mU. 1044P.mA.fC. A.fU.fU. A. 100% G.mC.mU. A. G.fC. A. G. G. A. G*A.mU. G.mU.Chl A*mU* G*mU* G* G. 14417 1497 1045 A.mC.mC.mU.mC. 1046P.mU. A.fU. A.fU. G.fU.  73% mC. A.mC. A.mU. G. G. A. G. G.mU*A.mU. A.Chl G*mC*mC* A*mU* C. 14418 1533 1047 A. A. 1048P.mU.fC.fC.fU. A. G.fU. 98% G.mU.mC.mC. G. G. A.mC.mU. A. G. G.A.mC.mU.mU*mU* A.Chl A*mU* A* G* U.   14419 1514 1049 mU. G. G.mU. G.1050 P.mU.fU.fU.fC.fU. G. 86% A.mU.mC. A. G. A. A.fU.fC. A.mC.mC.A. A.Chl A*mC*mU* G* G*mU* A.   14420 1534 1051 A. G.mU.mC.mC. 1052P.mU.fU.fC.fC.fU. A.  99% A.mC.mU. A. G. G. G.fU. G. G. A. A.ChlA.mC.mU*mU*mU* A*mU* A* G. 14421 943 1053 A.mC. G.mC.mC. 1054P.mA.fC.fC.fU.fC.fC.fU.  41% A. A. G. G. A. G. fU. G. G.mC. G.mU* A*G.mU.Chl G*mU* A*mC* U.   18570 2445 1055 mU. A.mU.mU.mU. 1056P.mU. A.fC. A.fC. A.  79% A.mU.mU. G.mU. A.fU. A. A. A.fU. A*G.mU. A.Chl A*fC*fU*fC* A* C. 18571 2445 1057 mU.mU. 1058P.mU. A.fC. A.fC. A.  75% A.mU.mU.mU. A.fU. A. A. A.fU. A*A.mU.mU. G.mU. A*fC*fU*fC* A* C. G.mU. A.Chl 18572 2083 1059A.mU. C. A. G.mU. 1060 P.mU.fU.fU.fU. A. A.fC.  47% G.mU.mU. A. A. A.A.fC.fU. G. A.fU* G* A* A.Chl A*fC*fC* A. 18573 2083 1061mC. A.mU.mC. A. 1062 P.mU.fU.fU.fU. A. A.fC.  17% G.mU. G.mU.mU.A.fC.fU. G. A.fU* G* A* A. A. A. A.Chl A*fC*fC* A. 18574 2544 1063A.mU. G. 1064 P.mU.fU.fC.fC.fU.fU. A.  59% G.mC.mU.mU. A.A. G.fC.fC. A. U*fC*fC* A. G. G. A. A.Chl A*fU* G* A. 18575 2544 1065G. A.mU. G. 1066 P.mU.fU.fC.fC.fU.fU. A. 141% G.mC.mU.mU. A.A. G.fC.fC. A. U*fC*fC* A. G. G. A. A.Chl A*fU* G* A. 18576 2137 1067mU.mU. G.mU. 1068 P.mU. A. A.fC. A. G. A.  77% G.mU.mU.mC.mU.A.fC. A.fC. A. A* G.mU.mU. A.Chl A*fC*fU*fU*fC* C. 18577 2137 1069mU.mU.mU. G.mU. 1070 P.mU. A. A.fC. A. G. A.  59% G.mU.mU.mC.mU.A.fC. A.fC. A. A* G.mU.mU. A.Chl A*fC*fU*fU*fC* C. 18578 2520 1071A. A. A.mU. 1072 P.mU. G. G.fC. A. A. A.  75% A.mC.mU.mU.mU.G.fU. A.fU.fU.fU* G* G.mC.mC. A.Chl G*fU*fC*fU* C. 18579 2520 1073mC. A. A. A.mU. 1074 P.mU. G. G.fC. A. A. A.  55% A.mC.mU.mU.mU.G.fU. A.fU.fU.fU* G* G.mC.mC. A.Chl G*fU*fC*fU* C. 18580 3183 1075mC.mU.mU. G.mC. 1076 P.mU.fU.fU. G.fU. A.  84% A.mC.mU. A.mC. A.G.fU. G.fC. A. A. A. A.Chl G*fU*fC* A* A* A* C. 18581 3183 1077A.mC.mU.mU. 1078 P.mU.fU.fU. G.fU. A.  80% G.mC. A.mC.mU.G.fU. G.fC. A. A. A.mC. A. A. A.Chl G*fU*fC* A* A* A* C. 18582 2267 1079G. A. 1080 P.mU. A.fC.fU. A. A.fU.  82% A.mU.mU.mU. A. A. A.mU.mU. A.A.fU.fU.fC*fU*fU*fC*fC* G.mU. A.Chl A* G. 18583 2267 1081 A. G. A. 1082P.mU. A.fC.fU. A. A.fU.  67% A.mU.mU.mU. A. A. A.mU.mU. A.A.fU.fU.fC*fU*fU*fC*fC* G.mU. A.Chl A* G. 18584 3184 1083 mU.mU. G.mC.1084 P.mU.fU.fU.fU. G.fU. A.  77% A.mC.mU. A.mC. A. G.fU. G.fC. A. A*A. A. A.Chl G*fU*fC* A* A* A. 18585 3184 1085 mC.mU.mU. G.mC. 1086P.mU.fU.fU.fU. G.fU. A.  59% A.mC.mU. A.mC. A. G.fU. G.fC. A. A*A. A. A.Chl G*fU*fC* A* A* A. 18586 2493 1087 A.mU. A. A. A. 1088P.mU.fC. A.fC.fC.fU.  84% A.mC. A. G. G.mU. G.fU.fU.fU.fU. G. A.ChlA.fU*fU*fU*fU*fC*fC* A. 18587 2493 1089 A. A.mU. A. A. A. 1090P.mU.fC. A.fC.fC.fU.  70% A.mC. A. G. G.mU. G.fU.fU.fU.fU. G. A.ChlA.fU*fU*fU*fU*fC*fC* A. 18588 2297 1091 G. A.mC. A. A.mC. 1092P.mU. G.fU.fU. G.fU.fU.  40% A. A.mC. A. A.mC. G.fU.fU. G.fU.fC* A.ChlG*fU*fU* G*fU* U. 18589 2046 1093 A.mU. G. 1094 P.mU.fU. G.fU.fU. A.fC. 39% C.mU.mU. G.mU. A. A. G.fC. A.fU*fC* A. A.mC. A. A.ChlA*fU*fC* G* U. 18590 2531 1095 mC. A. G. A. A. 1096 P.mU.fC. A.fU. G. A. 56% A.mC.mU.mC. G.fU.fU.fU.fC.fU. G* A.mU. G. A.Chl G*fC* A* A* A* G.18591 2389 1097 G.mU. A.mU.mU. 1098 P.mU. G.fC. A.fU. A.  64%G.mC.mU. A.mU. G.fC. A. A.fU. A.fC* A* G.mC. A.Chl G* A* A* A* A. 185922530 1099 mC.mC. A. G. A. A. 1100 P.mU. A.fU. G. A.  44% A.mC.mU.mC.G.fU.fU.fU.fC.fU. G. A.mU. A.Chl G*fC* A* A* A* G* U. 18593 2562 1101A.mC.mU.mC. A. 1102 P.mU. G.fC.fU.fC.  87% A. A.mC. G. A.G.fU.fU.fU. G. A. G.mC. A.Chl G.fU*fU*fC* A* A* G* U. 18594 2623 1103A.mU. A.mU. G. 1104 P.mU.fU.fC.fU.fC. G.  69% A.mC.mC. G. A. G.G.fU.fC. A.fU. A.fU* A* A. A.Chl A*fU* A* A* C. 18595 2032 1105mC. G. A.mC. G. 1106 P.mU.fU.fC. G.fU.fU.  55% A.mC. A. A.mC. G.G.fU.fC. G.fU.fC. A. A.Chl G*fU*fC* A*fU*fC* A. 18596 2809 1107G.mU. A. A. 1108 P.mU.fU.fC. A.fC.fU. G.  58% A.mC.mC. A. G.mU.G.fU.fU.fU. A.fC*fU* A* G. A. A.Chl A* A*fC* U. 18597 2798 1109mU.mU. G.mU.mC. 1110 P.mU.fC.fU. A. A.  38% A. G.mU.mU.mU.A.fC.fU. G. A.fC. A. A* A. G. A.Chl A* G* A* A*fC* C. 18598 2081 1111mU.mC. A.mU.mC. 1112 P.mU.fU. A. A.fC.  25% A. G.mU.A.fC.fU. G. A.fU. G. A* G.mU.mU. A. A.Chl A*fC*fC* A* A* G. 18599 25611113 A. A.mC.mU.mC. 1114 P.mU.fC.fU.fC.  57% A. A. A.mC. G. A. G.G.fU.fU.fU. G. A. A.Chl G.fU.fU*fC* A* A* G*fU* U. 18600 2296 1115mC. G. A.mC. A. 1116 P.mU.fU.fU. G.fU.fU.  69% A.mC. A. A.mC. A.G.fU.fU. G.fU.fC. A. A.Chl G*fU*fU* G*fU*fU* C. 18601 2034 1117A.mC. G. A.mC. A. 1118 P.mU.fC. A.fU.fC.  22% A.mC. G. A.mU. G.G.fU.fU. G.fU.fC. A.Chl G.fU*fC* G*fU*fC* A*fU. 18602 2681 1119 G.mC.mU.1120 P.mU.fU.fC.fC.fU.fU. A.  43% G.mC.mC.mU. A. A.G. G.fC. A. G.fC*fU* G* G. G. A. A.Chl A*fU* A* C. 18603 2190 1121A.mU.mU.mC.mU. 1122 P.mU. G. A. A. A.fU. 128% A.mC.G.fU. A. G. A. A.fU* A* A.mU.mU.mU.mC. A* G* G*fC* C. A.Chl 20604 20831123 mC. A.mU.mC. A. 1124 P.mU.fU.fU.fU. A. A.fC.  19% G.mU. G.mU.mU.A.fC.fU. G. A.mU* G* A. A. A. A.Chl A* A*mC*mC* A. 20605 2083 1125mC. A.mU.mC. A. 1126 P.mU.fU.fU.fU. A. A.fC.  20% G.mU. G.mU.mU.A.fC.fU. G. A.mU* G* A. A. A. A.Chl A* A*fC*mC* A. 20606 2083 1127mC. A.mU.mC. A. 1128 P.mU. U. U. U. A. A. C.  82% G.mU. G.mU.mU.A. C. U. G. A.mU* G* A. A. A. A.Chl A* A*mC*mC* A. 20607 2083 1129mC. A.mU.mC. A. 1130 P.mU.fU.fU.fU. A. A.fC.  59% G.mU. G.mU.mU.A.fC.fU. G. A. A. A. A.Chl A.fU*mG*mA*mA*fC*f C* A. 21722 2081 1131mU.mC. A.mU.mC. 1132 P.mU.fU. A. A.fC.  34% A. G.mU.A.fC.fU. G. A.fU. G. A* G.mU.mU. A. A.Chl A*mC*mC* A* A* G. 21723 20811133 mU.mC. A.mU.mC. 1134 P.mU.fU. A. A.fC.  53% A. G.mU.A.fC.fU. G. A.fU. G.mU.mU. A. A.Chl G.mA*mA*mC*mC*mA* mA* G. 21724 20811135 mU.mC. A.mU.mC. 1136 P.mU.fU. A. A.fC.  48% A. G.mU.A.fC.fU. G. A.mU. G.mU.mU. A. A.Chl G.mA*mA*mC*mC*mA* mA* G. 21725 20811137 mU.mC. A.mU.mC. 1138 P.mU.fU. A. A.fC.  45% A. G.mU.A.fC.fU. G. A.fU. G. A* G.mU.mU. A. A.Chl A*fC*fC*mA*mA* G. 21726 20811139 mU.mC. A.mU.mC. 1140 P.mU.fU. A. A.fC.  54% A. G.mU.A.fC.fU. G. A.fU. G.mU.mU. A. A.Chl G.mA*mA*fC*fC*mA* mA* G. 21727 20811141 mU.mC. A.mU.mC. 1142 P.mU.fU. A. A.fC.  29% A. G.mU.A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*fC*fC* A* A* G. TEG-Chl 217282081 1143 mU*mC* 1144 P.mU.fU. A. A.fC.  27% A.mU.mC. A.A.fC.fU. G. A.fU. G. A* G.mU. A*fC*fC* A* A* G. G.mU.mU*mA*mA. TEG-Chl21729 2081 1145 mU*mC*mA.mU.m 1146 P.mU.fU. A. A.fC.  30% C.mA.mG.mU.mG.A.fC.fU. G. A.fU. G. A* mU.mU*mA*mA. A*fC*fC* A* A* G. TEG-Chl 213752081 1147 mU.mC. A.mU.mC. 1148 P.mU.fU. A. A.fC.  29% A. G.mU.A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*mC*mC* A* A* G. TEG-Chl 213762081 1149 mU.mC. A.mU.mC. 1150 P.mU.fU. A. A.fC.  30% A. G.mU.A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*fC*fC*mA*m A* G. TEG-Chl 213772081 1151 mU.mC. A.mU.mC. 1152 P.mU.fU. A. A.fC.  37% A. G.mU.A.fC.fU. G. A.fU. G.mU.mU*mA*mA. G.mA*mA*fC*fC*mA* TEG-Chl mA* G. 213782081 1153 mU*mC*mA.mU.m 1154 P.mU.fU. A. A.fC.  32% C.mA.mG.mU.mG.A.fC.fU. G. A.fU. G. A* mU.mU*mA*mA. A*mC*mC* A* A* G. TEG-Chl 213792081 1155 mU*mC*mA.mU.m 1156 P.mU.fU. A. A.fC.  31% C.mA.mG.mU.mG.A.fC.fU. G. A.fU. G. A* mU.mU*mA*mA. A*fC*fC*mA*mA* G. TEG-Chl   213802081 1157 mU*mC*mA.mU.m 1158 P.mU.fU. A. A.fC.  39% C.mA.mG.mU.mG.A.fC.fU. G. A.fU. mU.mU*mA*mA. G.mA*mA*fC*fC*mA* TEG-Chl mA* G.   KeyChl = cholesterol with hydroxyprolinol linker TEG-chl = cholesterol withTEG linker m = 2′Ome f = 2′fluoro *= phosphorothioate ′linkage .=phosphodiester linkage

TABLE 7  TGFβ1 (Accession Number: NM_000660.3) % remaining Oligo StartSEQ ID SEQ ID expression Number Site NO Sense sequence NOAntisense sequence (1 uM A549) 14394 1194 1159 G.mC.mU. A. 1160P.mU.fU.fC.fC. A.fC.fC.  24% A.mU. G. G.mU. G. A.fU.fU. A. G.mC*G. A. A.Chl A*mC* G*mC* G* G. 14395 2006 1161 mU. G. A.mU.mC. 1162P.mG. A. G.fC. G.fC.  79% G.mU. G.mC. A.fC. G. A.mU.mC. G.mC.mU.mC.ChlA*mU* G*mU*mU* G* G. 14396 1389 1163 mC. A. 1164 P.mU.fC. G.fC.fC. A. G. 77% A.mU.mU.mC.mC. G. A. A.mU.mU. mU. G. G.mC. G. G*mU*mU* A.ChlG*mC*mU* G. 14397 1787 1165 A. G.mU. G. G. 1166 P.mU.fC. G.fU. G. G. n/aA.mU.mC.mC. A.fU.fC.fC. A.mC. G. A.Chl A.mC.mU*mU*mC*mC* A* G* C. 143981867 1167 mU. A.mC. A. 1168 P.mG. G. A.fC.fC.fU.fU.  82% G.mC. A. A. G.G.fC.fU. G.mU. G.mU.mC.mC.Chl A*mC*mU* G*mC* G* U. 14399 2002 1169A. A.mC. A.mU. G. 1170 P.mG.fC. A.fC. G. n/a A.mU.mC. G.mU.A.fU.fC. A.fU. G.mC.Chl G.mU.mU* G* G* A*mC* A* G. 14400 2003 1171A.mC. A.mU. G. 1172 P.mC. G.fC. A.fC. G. n/a A.mU.mC. G.mU.A.fU.fC. A.mU. G.mC. G.Chl G.mU*mU* G* G* A*mC* A. 1440 11869 1173mC. A. G.mC. A. A. 1174 P.mC. A. G. G.  82% G. A.fC.fC.fU.fU.G.mU.mC.mC.mU. G.mC.mU. G*mU* G.Chl A*mC*mU* G* C. 14402 2000 1175mC.mC. A. A.mC. 1176 P.mA.fC. G. A.fU.fC.  66% A.mU. G. A.mU.mC.A.fU. G.fU.mU. G. G* G.mU.Chl A*mC* A* G*mC* U. 14403 986 1177A. G.mC. G. G. A. 1178 P.mA.fU. G.fC.  78% A. G.mC. G.mC.G.fC.fU.fU.fC.fC. A.mU.Chl G.mC.mU*mU*mC* A*mC*mC* A. 14404 995 1179G.mC. A.mU.mC. 1180 P.mA.fU. G.  79% G. A. G. G.mC.mC.G.fC.fC.fU.fC. G. A.mU. A.mU.Chl G.mC* G*mC*mU*mU*mC* C. 14405 963 1181G. A.mC.mU. 1182 P.mC. A.fU. G.fU.fC. G.  80% A.mU.mC. G. A.mC. A.fU. A.A.mU. G.Chl G.mU.mC*mU*mU* G*mC* A* G. 14406 955 1183 A.mC.mC.mU. 1184P.mU. A. G.fU.fC.fU.fU.  88% G.mC. A. A. G. G.fC. A. G. G.mU* G*A.mC.mU. A.Chl G* A*mU* A* G. 14407 1721 1185 G.mC.mU.mC.mC. 1186P.mU.fU.fC.fU.fC.fC. n/a A.mC. G. G. A. G. A. G.fU. G. G. A. A.ChlG.mC*mU* G* A* A* G* C. 18454 1246 1187 mC. A.mC. A. G.mC. 1188P.mU. A.fU. A.fU. A.fU.  58% A.mU. A.mU. A.mU. G.fC.fU. G.fU. G*fU*A.Chl G*fU* A*fC* U. 18455 1248 1189 mC. A. G.mC. 1190P.mU. A.fU. A.fU. A.fU.  87% A.mU. A.mU. A.mU. A.fU. G.fC.fU. G*fU*A.mU. A.Chl G*fU* G*fU* A. 18456 1755 1191 G.mU. A.mC. 1192P.mU. A. A. G.fU.fC. A. 107% A.mU.mU. G. A.fU. G.fU. A.fC* A*A.mC.mU.mU. G*fC*fU* G* C. A.Chl 18457 1755 1193 mU. G.mU. A.mC. 1194P.mU. A. A. G.fU.fC. A.  77% A.mU.mU. G. A.fU. G.fU. A.fC* A*A.mC.mU.mU. G*fC*fU* G* C. A.Chl 18458 1708 1195 A. A.mC.mU. 1196P.mU. G. A. A. G.fC. A.  75% A.mU.mU. A.fU. A. G.fU.fU* G*G.mC.mU.mU.mC. G*fU* G*fU* C. A.Chl 18459 1708 1197 mC. A. A.mC.mU. 1198P.mU. G. A. A. G.fC. A.  73% A.mU.mU. A.fU. A. G.fU.fU* G*G.mC.mU.mU.mC. G*fU* G*fU* C. A.Chl 18460 1250 1199 G.mC. A.mU. 1200P.mU. A.fC. A.fU. A.fU. n/a A.mU. A.mU. A.mU. A.fU. A.fU. G.fC*fU*G.mU. A.Chl G*fU* G*fU* G. 18461 1754 1201 mU. G.mU. A.mC. 1202P.mU. A. G.fU.fC. A.  91% A.mU.mU. G. A.fU. G.fU. A.fC. A*A.mC.mU. A.Chl G*fC*fU* G*fC* C. 18462 1754 1203 mC.mU. G.mU. 1204P.mU. A. G.fU.fC. A.  92% A.mC. A.mU.mU. G. A.fU. G.fU. A.fC. A*A.mC.mU. A.Chl G*fC*fU* G*fC* C. 18463 1249 1205 A. G.mC. A.mU. 1206P.mU.fC. A.fU. A.fU. n/a A.mU. A.mU. A.mU. A.fU. A.fU. G.fC.fU* G. A.ChlG*fU* G*fU* G* U. 18464 1383 1207 mC. A. G.mC. A. 1208P.mU. G. A. A.fU.fU.  77% A.mC. A. G.fU.fU. G.fC.fU. G*fU* A.mU.mU.mC.A*fU*fU*fU* C. A.Chl 18465 1251 1209 mC. A.mU. A.mU. 1210P.mU. A. A.fC. A.fU.  84% A.mU. A.mU. A.fU. A.fU. A.fU. G.mU.mU. A.ChlG*fC*fU* G*fU* G* U. 18466 1713 1211 mU.mU. 1212 P.mU. G. A. G.fC.fU. G.n/a G.mC.mU.mU.mC. A. A. G.fC. A. A*fU* A* A. G.mC.mU.mC. G*fU*fU* G.A.Chl 18467 1713 1213 A.mU.mU. 1214 P.mU. G. A. G.fC.fU. G.  83%G.mC.mU.mU.mC. A. A. G.fC. A. A*fU* A* A. G.mC.mU.mC. G*fU*fU* G. A.Chl18468 1247 1215 A.mC. A. G.mC. 1216 P.mU.fU. A.fU. A.fU.  96%A.mU. A.mU. A.mU. A.fU. G.fC.fU. G.fU* A. A.Chl G*fU* G*fU* A* C. 184691712 1217 A.mU.mU. 1218 P.mU. A. G.fC.fU. G. A.  90% G.mC.mU.mU.mC.A. G.fC. A. A.fU* A* A. G.mC.mU. A.Chl G*fU*fU* G* G. 18470 1712 1219mU. A.mU.mU. 1220 P.mU. A. G.fC.fU. G. A.  98% G.mC.mU.mU.mC.A. G.fC. A. A.fU* A* A. G.mC.mU. A.Chl G*fU*fU* G* G. 18471 1212 1221mC. A. A. 1222 P.mU.fU. G.fC.fU.fU. G. n/a G.mU.mU.mC. A. A.A. A.fC.fU.fU. G*fU*fC* G.mC. A. A.Chl A*fU* A* G. 18472 1222 1223mC. A. G. A. G.mU. 1224 P.mU. G.fU. G.fU. G.fU.  45% A.mC. A.mC. A.mC.A.fC.fU.fC.fU. G* A.Chl C*fU*fU* G* A* A. 18473 1228 1225A.mC. A.mC. A.mC. 1226 P.mU.fU. A.fU. G.fC.fU.  36% A. G.mC. A.mU. A.G.fU. G.fU. G.fU* A.Chl A*fC*fU*fC*fU* G. 18474 1233 1227 mC. A. G.mC.1228 P.mU. A.fU. A.fU. A.fU.  68% A.mU. A.mU. A.mU. A.fU. G.fC.fU. G*fU*A.mU. A.Chl G*fU* G*fU* A. 18475 1218 1229 mU.mC. A. A. 1230P.mU.fU. A.fC.fU.fC.fU.  64% G.mC. A. G. A. G.fC.fU.fU. G. A*G.mU. A. A.Chl A*fC*fU*fU* G* U. 18476 1235 1231 A. G.mC. A.mU. 1232P.mU.fC. A.fU. A.fU.  78% A.mU. A.mU. A.mU. A.fU. A.fU. G.fC.fU*G. A.Chl G*fU* G*fU* G* U. 18477 1225 1233 A. G. A. G.mU. 1234P.mU.fU. G.fU. G.fU.  92% A.mC. A.mC. A.mC. G.fU. A.fC.fU.fC.fU*A. A.Chl G*fC*fU*fU* G* A. 18478 1221 1235 A. A. G.mC. A. G. A. 1236P.mU.fU. G.fU. 103% G.mU. A.mC. A. A.fC.fU.fC.fU. A.ChlG.fC.fU.fU* G* A* A*fC*fU* U. 18479 1244 1237 mU.mU.mC. A. 1238P.mU.fU. G. A.fU. G.fU.  84% A.mC. A.mC. G.fU.fU. G. A. A* G* A*A.mU.mC. A. A.Chl A*fC* A* U. 18480 1224 1239 A. G.mC. A. G. A. 1240P.mU. G.fU. G.fU.  37% G.mU. A.mC. A.mC. A.fC.fU.fC.fU. A.ChlG.fC.fU*fU* G* A* A*fC* U. 18481 1242 1241 A.mU. A.mU. 1242P.mU. A. A. G. A. A.fC.  62% A.mU. A.fU. A.fU. A.fU* A*fU*G.mU.mU.mC.mU. G*fC*fU* G. mU. A.Chl 18482 1213 1243 G. A.mC. A. A. 1244P.mU.fC.fU.fU. G. A.  47% G.mU.mU.mC. A. A. A.fC.fU.fU. G.fU.fC*G. A.Chl A*fU* A* G* A* U. 18483 1760 1245 mU.mU. A. A. A. G. 1246P.mU.fC.fU.fC.fC.  69% A.mU. G. G. A. G. A.fU.fC.fU.fU.fU. A. A.ChlA*fU* G* G* G* G* C. 18484 1211 1247 mC.mU. A.mU. G. 1248P.mU. A. A.fC.fU.fU. n/a A.mC. A. A. G.fU.fC. A.fU. A. G* G.mU.mU. A.ChlA*fU*fU*fU*fC* G. 19411 1212 1249 mC. A. A.mC. G. A. 1250 P.mU.fU. A. G. 52% A. A.mU.mC.mU. A. A.fU.fU.fU.fC. G.fU.fU. A.ChlG*fU* G* G* G*fU*fU. 19412 1222 1251 mU. A.mU. G. 1252P.mU. G. A. A.fC.fU.fU.  51% A.mC. A. A. G.fU.fC. A.fU. A* G*G.mU.mU.mC. A*fU*fU*fU*fC. A.Chl 19413 1228 1253 A. A. 1254P.mU.fC.fU. G.fC.fU.fU. n/a G.mU.mU.mC. A. A. G. A. A.fC.fU.fU*G.mC. A. G. A.Chl G*fU*fC* A*fU* A. 19414 1233 1255 mC. A. A. G.mC. A.1256 P.mU. G.fU  41% G. A. G.mU. A.mC. A.fC.fU.fC.fU. A.ChlG.fC.fU.fU. G* A* A*fC*fU*fU* G. 19415 1218 1257 A. A.mU.mC.mU. 1258P.mU.fU.fU. G.fU.fC. 104% A.mU. G. A.mC. A. A.fU. A. G. A. A.ChlA.fU.fU*fU*fC* G*fU*fU* G. 19416 1244 1259 mC. A.mC. A.mC. A. 1260P.mU. A.fU. A.fU.  31% G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.ChlG*fU* A*fC*fU*fC*fU. 19417 655 1261 G. A. A. A.mU. 1262P.mU.fU.fU. G.fC.fU. n/a A.mU. A. G.mC. A. A.fU. A.fU.fU.fU.fC*fU*A. A.Chl G* G*fU* A* G. 19418 644 1263 G. A. 1264P.mU.fC.fU. G. G.fU. A. n/a A.mC.mU.mC.mU. G. A. G.fU.fU.fC*fU*A.mC.mC. A. G. A*fC* G*fU* G. A.Chl 19419 819 1265 G.mC. A. A. A. G.1266 P.mU.fC. A.fU.fU. n/a A.mU. A. A.mU. G. A.fU.fC.fU.fU.fU. A.ChlG.fC*fU* G*fU*fC* A* C. 19420 645 1267 A. 1268 P.mU.fU.fC.fU. G. G.fU.n/a A.mC.mU.mC.mU. A. G. A. G.fU.fU*fC*fU* A.mC.mC. A. G. A. A*fC* G* U.A.Chl 19421 646 1269 A.mC.mU.mC.mU. 1270 P.mU.fU.fU.fC.fU. G. n/aA.mC.mC. A. G. A. G.fU. A. G. A. A. A.Chl G.fU*fU*fC*fU* A*fC* G. 19422816 1271 A.mC. A. G.mC. A. 1272 P.mU.fU. n/a A. A. G. A.mU. A.A.fU.fC.fU.fU.fU. A.Chl G.fC.fU. G.fU*fC* A*fC* A* A* G. 19423 495 1273mC. A. 1274 P.mU.fU. G.fU.fC. A.fU. n/a A.mU.mC.mU. A. G. A.fU.fU. G*fC*A.mU. G. A.mC. A. G*fU*fU* G* U. A.Chl 19424 614 1275 A. G. 1276P.mU.fU. G. A.fC.fU.fU. n/a A.mU.mU.mC. A. A. G. A. A.fU.fC.fU*fC*fU*G.mU.mC. A. A.Chl G*fC* A* G. 19425 627 1277 mC.mU. G.mU. G. 1278P.mU. G.fU.fU. n/a G. A. G.mC. A. G.fC.fU.fC.fC. A.fC. A. A.mC. A.ChlG*fU*fU* G* A*fC* U. 19426 814 1279 mU. G. A.mC. A. 1280P.mU.fU.fC.fU.fU.fU. n/a G.mC. A. A. A. G. A. G.fC.fU. G.fU.fC. A*fC*A.Chl A* A* G* A* G. 19427 501 1281 A.mU. G. A.mC. A. 1282 P.mU.fU. G.n/a A. A. A.mC.mC. A. G.fU.fU.fU.fU. G.fU.fC. A.Chl A.fU* A* G* A*fU*fU*G. 19428 613 1283 G. A. G. 1284 P.mU. G. A.fC.fU.fU. G. n/aA.mU.mU.mC. A. A. A. A.fU.fC.fU.fC*fU* G.mU.mC. A.Chl G*fC* A* G* G.21240 1244 1285 mC. A.mC. A.mC. A. 1286 P.mU. A.fU. A.fU. 0.875G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*mU* A*mC*mU*mC* U. 212411244 1287 mC. A.mC. A.mC. A. 1288 P.mU. A.fU. A.fU. 0.88 G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*mU*mA*mC*mU*m C* U. 212421244 1289 mC. A.mC. A.mC. A. 1290 P.mU. A.fU. A.fU. 0.635G.mC. A.mU. A.mU. G.fC.fU. G.fU. A.Chl G.fU.mG*mU*mA*mC* mU*mC* U. 212431244 1291 mC. A.mC. A.mC. A. 1292 P.mU. A.fU. A.fU. 0.32 G.mC. A.mU. A.mU. G.fC.fU. G.fU. A.Chl G.fU.mG*fU*mA*fC*m U*fC* U. 212441244 1293 mC. A.mC. A.mC. A. 1294 P.mU. A.fU. A.fU. 0.36 G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*fU* A*fC*mU*mC* U. 212451244 1295 mC. A.mC. A.mC. A. 1296 P.mU. A.fU. A.fU. 0.265 G.mC. A.mU.G.fC.fU. G.fU. G.fU. A*mU*mA.TEG-Chl G*fU* A*fC*fU*fC*fU. 21246 12441297 mC*mA*mC. A.mC. 1298 P.mU. A.fU. A.fU. 0.334 A. G.mC. A.mU.G.fC.fU. G.fU. G.fU. A*mU*mA.TEG-Chl G*fU* A*fC*fU*fC*fU. 21247 12441299 mC*mA*mC.mA.m 1300 P.mU. A.fU. A.fU. 0.29  C.mA.mG.mC.mA.G.fC.fU. G.fU. G.fU. mU.mA*mU*mA.T G*fU* A*fC*fU*fC*fU. EG-Chl 21248 6141301 mA. G. 1302 P.mU.fU. G. A.fC.fU.fU. n/a A.mU.mU.mC. A. A.G. A. A.fU.fC.fU*fC*fU* G.mU.mC*mA*mA. G*fC*fU* U. TEG-Chl 1303 130420608 1244 1305 mC. A.mC. A.mC. A. 1306 P.mU. A.fU. A.fU.  79%G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.mU. A.Chl G*mU* A*mC*mU*mC* U. 206091244 1307 mC. A.mC. A.mC. A. 1308 P.mU. A.fU. A.fU.  60%G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.mU. A.Chl G*fU* A*mC*fU*mC* U. 206101244 1309 mC. A.mC. A.mC. A. 1310 P.mU. A. U. A. U. G. C.  93%G.mC. A.mU. A.mU. U. G. U. G.mU. G*mU* A.Chl A*mC*mU*mC* U. 20611 12441311 mC. A.mC. A.mC. A. 1312 P.mU. A.fU. A.fU. n/a G.mC. A.mU. A.mU.G.fC.fU. G.fU. A.Chl G.mU.mG*mU*mA*mC *mU*mC* U. 21374 614 1313mC*mA*mC.mA.m 1314 P.mU. A.fU. A.fU.  24% C.mA.mG.mC.mA. G.fC.fU. G.fU.mU.mA*mU*mA.T G.fU.mG*fU*mA*fC*m EG-Chl U*fC* U. Key Chl = cholesterolwith hydroxyprolinol linker TEG-chl =c holesterol with TEG linker m =2′Ome f = 2′fluoro *= phosphorothioate ′linkage .= phosphodiesterlinkage

TABLE 8 Examples of VEGF (Accession No. NM_001171623.1) sd-rxRNA sequences% remaining mRNA expression (qPCR, 1 uM Oligo Gene Ref SEQ SEQ sd-rxRNA,ID Region Pos ID Sense sequence ID Antisense sequence Hek293) 19850 CDS1389 1315 GAUGAGCUUCCUA 1316 UAGGAAGCUCAUCUCUCCU 161% 19851 3′UTR 18531317 AGAACAGUCCUUA 1318 UAAGGACUGUUCUGUCGAU  38% 19852 3′UTR 1854 1319GAACAGUCCUUAA 1320 UUAAGGACUGUUCUGUCGA 124% 19853 3′UTR 1857 1321CAGUCCUUAAUCA 1322 UGAUUAAGGACUGUUCUGU 124% 19854 3′UTR 1859 1323GUCCUUAAUCCAA 1324 UUGGAUUAAGGACUGUUCU n/a 19855 3′UTR 1863 1325UUAAUCCAGAAAA 1326 UUUUCUGGAUUAAGGACUG 120% 19856 3′UTR 2183 1327UGUUAUUGGUGUA 1328 UACACCAAUAACAUUAGCA 114% 19857 3′UTR 2790 1329UUGAAACCACUAA 1330 UUAGUGGUUUCAAUGGUGU 136% 19858 3′UTR 2931 1331GAGAAAAGAGAAA 1332 UUUCUCUUUUCUCUGCCUC 103% 19859 3′UTR 2932 1333AGAAAAGAGAAAA 1334 UUUUCUCUUUUCUCUGCCU 106% 19860 3′UTR 2933 1335GAAAAGAGAAAGA 1336 UCUUUCUCUUUUCUCUGCC 115% 19861 3′UTR 3199 1337ACACUCAGCUCUA 1338 UAGAGCUGAGUGUUAGCAA 123% 19862 3′UTR 3252 1339AAAUAAGGUUUCA 1340 UGAAACCUUAUUUCAAAGG 131% 19863 3′UTR 3427 1341AAUCUCUCUCCUA 1342 UAGGAGAGAGAUUUAGUAU 103% 19864 3′UTR 3429 1343UCUCUCUCCUUUA 1344 UAAAGGAGAGAGAUUUAGU 136% 19865 3′UTR 3430 1345CUCUCUCCUUUUA 1346 UAAAAGGAGAGAGAUUUAG 130% 19866 3′UTR 3471 1347AUUGGUGCUACUA 1348 UAGUAGCACCAAUAAAUAA 125% 19867 3′UTR 3476 1349UGCUACUGUUUAA 1350 UUAAACAGUAGCACCAAUA  93% 19868 3′UTR 1852 1351CAGAACAGUCCUA 1352 UAGGACUGUUCUGUCGAUG  83% 19869 CDS 1343 1353UGCAGAUUAUGCA 1354 UGCAUAAUCUGCAUGGUGA n/a 19870 CDS 1346 1355GAUUAUGCGGAUA 1356 UAUCCGCAUAAUCUGCAUG n/a 19871 CDS 1352 1357UGCGGAUCAAACA 1358 UGUUUGAUCCGCAUAAUCU  74% 19872 3′UTR 1985 1359GGAUUCGCCAUUA 1360 UAAUGGCGAAUCCAAUUCC n/a 19873 3′UTR 2210 1361UUGACUGCUGUGA 1362 UCACAGCAGUCAAAUACAU n/a 19874 3′UTR 2447 1363CAGAAAGACAGAA 1364 UUCUGUCUUUCUGUCCGUC n/a 19875 3′UTR 2792 1365GAAACCACUAGUA 1366 UACUAGUGGUUUCAAUGGU n/a 19876 3′UTR 2794 1367AACCACUAGUUCA 1368 UGAACUAGUGGUUUCAAUG n/a 19877 3′UTR 3072 1369UAUCUUUUGCUCA 1370 UGAGCAAAAGAUACAUCUC n/a 19878 3′UTR 3073 1371AUCUUUUGCUCUA 1372 UAGAGCAAAAGAUACAUCU n/a 19879 3′UTR 3162 1373UCACUAGCUUAUA 1374 UAUAAGCUAGUGACUGUCA n/a 19880 3′UTR 3163 1375CACUAGCUUAUCA 1376 UGAUAAGCUAGUGACUGUC n/a

TABLE 9  Examples of selected VEGF rxRNAori Sequences Oligo IDStart Site 25 mer Sense Sequence 25 mer Anti-sense sequence 18760 18535′-AUCACCAUCGACAGAACAGUCCUUA 5′-UAAGGACUGUUCUGUCGAUGGUGAU(SEQ ID NO: 13) (SEQ ID NO: 1377) 18886 13525′-CCAUGCAGAUUAUGCGGAUCAAACA 5′-UGUUUGAUCCGCAUAAUCUGCAUGG(SEQ ID NO: 28) (SEQ ID NO: 1378)

TABLE 10  Optimized VEGF sd-rxRNA Sequences With Increased StabilityDuplex Oligo ID SEQ ID NO 19851 19790 1379A. G. A. A.mC. A. G.mU.mC.mC.mU.mU. A.Chl 19791 1380P.mU. A. A. G. G. A.fC.fU. G.fU.fU.fC.fU* G*fU*fC* G* A* U DescriptionSS 3′ Ome block 1381 A.G.A.A.mC.A.G.mU.mC.mC.mU*mU*mA-TEG-ChlComplete Ome 1382 mA.mG.mA.mA.mC.mA.mG.mU.mC.mC.mU*mU*mA-TEG-Chl 3′and 5′ Ome 1383 mA.mG.A.A.mC.A.G.mU.mC.mC.mU*mU*mA-TEG-Chl blockAS - no > 3 Pos 5 2′Ome G 1384P.mU.A.A.G.mG.A.fC.fU.G.fU.fU.fC.fU*G*fU*fC*G*A*U 2′OH Pos 4 2′Ome G1385 P.mU.A.A.mG.G.A.fC.fU.G.fU.fU.fC.fU*G*fU*fC*G*A*U Pos 3 2′Ome A1386 P.mU.A.mA.G.G.A.fC.fU.G.fU.fU.fC.fU*G*fU*fC*G*A*U Pos 4 2′F G  1387 P.mU.A.A.fG.G.A.fC.fU.G.fU.fU.fC.fU*G*fU*fC*G*A*U StabilizingNo 2′OH 3′ tail 1388P.mU.A.A.mG.G.A.fC.fU.G.fU.fU.fC.fU*mG*fU*fC*mG*mA*U 3′ end (no(1) 2′OH 3′ tail 1389P.mU.A.A.mG.G.A.fC.fU.G.fU.fU.fC.fU*G*fU*fC*mG*mA*U 2′OH No 2′OH 3′ tail1390 P.mU.A.A.fG.G.A.fC.fU.G.fU.fU.fC.fU*mG*fU*fC*mG*mA*U (1) 2′OH 3′tail 1391 P.mU.A.A.fG.G.A.fC.fU.G.fU.fU.fC.fU*G*fU*fC*mG*mA*U No 2′OH 3′tail 1392 P.mU.A.A.fG.G.A.fC.fU.G.fU.fU.fC.fU*fG*fU*fC*mG*mA*U5 Methyl C 1393 P.mY.A.A.fG.G.A.fX.fY.G.fY.fY.fX.fU*G*fY*fX*mG*mA*Uand U 1394 P.mY.A.A.fG.G.A.fX.fY.G.fY.fY.fX.fU*mG*fY*fX*mG*mA*U 1395P.mY.A.A.mG.G.A.fX.fY.G.fY.fY.fX.fU*G*fY*fX*mG*mA*U 1396P.mY.A.A.mG.G.A.fX.fY.G.fY.fY.fX.fU*mG*fY*fX*mG*mA*U 19871 19830 1397mU. G.mC. G. G. A.mU.mC. A. A. A.mC. A.Chl 19831 1398P.mU. G.fU.fU.fU. G. A.fU.fC.fC. G.fC. A*fU* A* A*fU*fC* U Key Chl =cholesterol with hydroxyprolinol linker TEG-chl = cholesterol with TEGlinker m = 2′Ome f = 2′fluoro *= phosphorothioate llinkage .=phosphodiester linkage

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety. This applicationincorporates by reference the entire contents, including all thedrawings and all parts of the specification (including sequence listingor amino acid/polynucleotide sequences) of PCT Publication No.WO2010/033247 (Application No. PCT/US2009/005247), filed on Sep. 22,2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS” and PCTPublication No. WO2009/102427 (Application No. PCT/US2009/000852), filedon Feb. 11, 2009, and entitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USESTHEREOF.”

What is claimed is:
 1. A method for delivering a nucleic acid to an eyeof a subject in need thereof, comprising administering to the eye of thesubject an sd-rxRNA, in an effective amount to promote RNA interferenceby the sd-rxRNA in the eye.
 2. The method of claim 1, wherein theadministration of the sd-rxRNA is intravitreal.
 3. The method of claim1, wherein the method is a method for treating an ocular disorder,optionally wherein the ocular disorder is selected from the groupconsisting of: vascular leakage, neovascularization, age-related maculardegeneration (AMD), choroidal neovascularization (wet AMD), geographicatrophy (advanced dry AMD), early-to-intermediate dry AMD, post surgicalcystoid macular edema (CME), nonproliferative diabetic retinopathy(NPDR), diabetic macular edema (DME), macular edema secondary to retinalvein occlusion (RVO), proliferative diabetic retinopathy (PDR),glaucoma, neovascular glaucoma (NVG), retinopathy of prematurity (ROP),fibroproliferative retinal disease, proliferative vitreoretinopathy(PVR), epiretinal membranes/vitreomacular adhesions, retinaldegenerative disease, retinitis pigmentosa, retinal vascular occlusivedisorders, retinal vein occlusion, retinal artery occlusion,retinoblastoma, trabeculectomy failure due to scarring, and uveitis.4.-6. (canceled)
 7. The method of claim 3, wherein the ocular disorderis proliferative vitreoretinopathy (PVR).
 8. (canceled)
 9. The method ofclaim 1, wherein the sd-rxRNA is directed against a gene encoding for aprotein selected from the group consisting of: VEGF, MAP4K4, PDGF-B,SDF-1, IGTA5, ANG2, CTGF, HIF-1α, mTOR, SDF-1, PDGF-B, SPP1, PTGS2(COX-2), TGFβ1, TGFβ2, complement factors 3 and 5, PDGFRα, PPIB, andmyc, or a combination thereof.
 10. The method of claim 9, wherein thesd-rxRNA is directed against a gene encoding for VEGF.
 11. The method ofclaim 9, wherein the sd-rxRNA is directed against a gene encoding forCTGF.
 12. The method of claim 1, wherein two or more different sd-rxRNAsthat are directed against genes encoding for two or more differentproteins are both administered to the eye of the subject.
 13. The methodof claim 12, wherein the sd-rxRNAs comprise sd-rxRNAs directed againstVEGF and CTGF.
 14. (canceled)
 15. The method of claim 10, wherein thesd-rxRNA is directed against a sequence selected from the sequenceswithin Table 2, optionally wherein the sd-rxRNA is directed against asequence comprising at least 12 contiguous nucleotides of a sequenceselected from the sequences within Table
 2. 16. (canceled)
 17. Themethod of claim 1, wherein the sd-rxRNA comprises at least 12 contiguousnucleotides of a sequence selected from the sequences within Tables 3-8or
 10. 18. The method of claim 10, wherein the sense strand of thesd-rxRNA comprises at least 12 contiguous nucleotides of the sequence ofSEQ ID NO:1317 or SEQ ID NO:1357 and/or wherein the antisense strand ofthe sd-rxRNA comprises at least 12 contiguous nucleotides of thesequence of SEQ ID NO:1318 or SEQ ID NO:1358.
 19. (canceled)
 20. Themethod of claim 10, wherein the sense strand of the sd-rxRNA comprisesSEQ ID NO:1317 and the antisense strand of the sd-rxRNA comprises SEQ IDNO:1318, the sense strand of the sd-rxRNA comprises SEQ ID NO:1357 andthe antisense strand of the sd-rxRNA comprises SEQ ID NO:1358, the sensestrand of the sd-rxRNA comprises SEQ ID NO:1379 and the antisense strandof the sd-rxRNA comprises SEQ ID NO:1380, or the sense strand of thesd-rxRNA comprises SEQ ID NO:1397 and the antisense strand of thesd-rxRNA comprises SEQ ID NO:1398. 21.-24. (canceled)
 25. The method ofclaim 11, wherein the sense strand of the sd-rxRNA comprises at least 12contiguous nucleotides of the sequence of SEQ ID NO:947 or SEQ ID NO:963and/or wherein the antisense strand of the sd-rxRNA comprises at least12 contiguous nucleotides of the sequence of SEQ ID NO:948 or SEQ IDNO:964.
 26. The method of claim 1, wherein the sd-rxRNA ishydrophobically modified, optionally wherein the sd-rxRNA is linked toone or more hydrophobic conjugates and/or the sd-rxRNA includes at leastone 5-methyl C or U modifications. 27.-28. (canceled)
 29. A method fordelivering a nucleic acid to an eye of a subject in need thereof,comprising administering to the eye of the subject an rxRNAori, in aneffective amount to promote RNA interference by the rxRNAori in the eye.30.-33. (canceled)
 34. An sd-rxRNA that is directed against a sequenceselected from the sequences within Table 2, optionally wherein thesd-rxRNA is directed against a sequence comprising at least 12contiguous nucleotides of a sequence selected from the sequences withinTable 2, or that comprises at least 12 contiguous nucleotides of asequence selected from the sequences within Tables 3-8 or
 10. 35.-36.(canceled)
 37. The sd-rxRNA of claim 34, wherein the sense strand of thesd-rxRNA comprises at least 12 contiguous nucleotides of the sequence ofSEQ ID NO:1317, 1357, 1379 or 1397 and/or wherein the antisense strandof the sd-rxRNA comprises at least 12 contiguous nucleotides of thesequence of SEQ ID NO:1318, 1358, 1380 or
 1398. 38. (canceled)
 39. Thesd-rxRNA of claim 34, wherein the sense strand of the sd-rxRNA comprisesSEQ ID NO:1317 and the antisense strand of the sd-rxRNA comprises SEQ IDNO:1318, the sense strand of the sd-rxRNA comprises SEQ ID NO:1357 andthe antisense strand of the sd-rxRNA comprises SEQ ID NO:1358, the sensestrand of the sd-rxRNA comprises SEQ ID NO:1379 and the antisense strandof the sd-rxRNA comprises SEQ ID NO:1380 or the sense strand of thesd-rxRNA comprises SEQ ID NO:1397 and the antisense strand of thesd-rxRNA comprises SEQ ID NO:
 1398. 40.-42. (canceled)
 43. The sd-rxRNAof claim 34, wherein the sd-rxRNA is hydrophobically modified,optionally wherein the sd-rxRNA is linked to one or more hydrophobicconjugates and/or the sd-rxRNA includes at least one 5-methyl C or Umodifications. 44.-45. (canceled)
 46. A composition comprising ansd-rxRNA of claim
 34. 47. The composition of claim 46, wherein thecomposition further comprises an sd-rxRNA that is directed against agene encoding for a protein other than VEGF, optionally wherein thecomposition comprises an sd-rxRNA that is directed against a geneencoding for CTGF and/or PTGS2 (COX-2). 48.-55. (canceled)